Methods for detecting the presence of mutant mammalian presenilin protein

ABSTRACT

The present invention describes the identification, isolation, cloning, and determination of the Alzheimer Related Membrane Protein (ARMP) gene on chromosome 14 and a related gene, E5-1, on chromosome 1. Normal and mutant copies of both genes are presented. Transcripts and products of these genes are useful in detecting and diagnosing Alzheimer&#39;s disease, developing therapeutics for treatment of Alzheimer&#39;s disease, as well as the isolation and manufacture of the protein and the construction of transgenic animals expressing the mutant genes.

RELATED APPLICATIONS

The application is a divisional of U.S. application Ser. No. 11/070,405,filed Feb. 24, 2005, which is a continuation of U.S. application Ser.No. 09/689,159, filed Oct. 12, 2000, now U.S. Pat. No. 6,998,467, whichis a divisional of U.S. application Ser. No. 08/509,359, filed Jul. 31,1995, now abandoned, which is a continuation-in-part of U.S. patentapplication Ser. No. 08/496,841, filed Jun. 28, 1995, now U.S. Pat. No.6,210,919, which is a continuation-in-part of U.S. patent applicationSer. No. 08/431,048, filed Apr. 28, 1995, now U.S. Pat. No. 6,531,586.

FIELD OF THE INVENTION

The present invention relates generally to the field of neurological andphysiological dysfunctions associated with Alzheimer's Disease. Moreparticularly, the invention is concerned with the identification,isolation and cloning of the gene which when mutated is associated withAlzheimer's Disease as well as its transcript, gene products andassociated sequence information and neighbouring genes. The presentinvention also relates to methods of diagnosing for and detection ofcarriers of the gene, Alzheimer's Disease diagnosis, gene therapy usingrecombinant technologies and therapy using the information derived fromthe DNA, protein, and the metabolic function of the protein.

BACKGROUND OF THE INVENTION

In order to facilitate reference to various journal articles, a listingof the articles is provided at the end of this specification.

Alzheimer's Disease (AD) is a degenerative disorder of the human centralnervous system characterized by progressive memory impairment andcognitive and intellectual decline during mid to late adult life(Katzman, 1986). The disease is accompanied by a constellation ofprincipal amongst which neuropathologic features are the presence ofextracellular amlyoid or senile plaques and the neurofibrillarydegeneration of neurons. The etiology of this disease is complex,although in some families it appears to be inherited as an autosomaldominant trait. However, even among these inherited forms of AD, thereare at least three different genes which confer inherited susceptibilityto this disease (St. George-Hyslop et al., 1990). The _(e)4 (Cys112Arg)allelic polymorphism of the Apolipoprotein E (AopE) gene has beenassociated with AD in a significant proportion of cases with onset latein life (Saunders et al., 1993; Strittmatter et al., 1993). Similarly, avery small proportion of familial cases with onset before age 65 yearshave been associated with mutations in the B amyloid precursor protein(APP) gene (Chartier-Harlin et al., 1991; Goate et al., 1991; Murrell etal., 1991; Karlinsky et al., 1992; Mullan et al., 1992). A third locus(AD3) associated with a larger proportion of cases with early onset ADhas recently been mapped to chromosome 14q24.3 (Schellenberg et al.,1992; St. George-Hyslop et al., 1992; Van Broeckhoven et al., 1992).

Although chromosome 14q carries several genes which could be regarded ascandidate genes for the site of mutations associated with AD3 (e.g.cF0S, alpha—1—antichymotrypsin, and cathepsin G), most of thesecandidate genes have been excluded on the basis of their physicallocation outside the AD3 region and/or the absence of mutations in theirrespective open reading frames (Schellenberg, G D et al., 1992; VanBroeckhoven, C et al., 1992; Rogaev et al., 1993; Wong et al., 1993).

There have been several developments and commercial directions inrespect of treatment of Alzheimer's disease and diagnosis thereof.Published PCT application WO 94 23049 describes transfection of highmolecular weight YAC DNA into specific mouse cells. This method is usedto analyze large gene complexes, for example the transgenic mice mayhave increased amyloid precursor protein gene dosage, which mimics thetrisomic condition that prevails in Downs Syndrome and the generation ofanimal models with β-amyloidosis prevalent in individuals withAlzheimer's Disease. Published international application WO 94 00569describes transgenic non-human animals harbouring large trans genes suchas the trans gene comprising a human amyloid precursor protein gene.Such animal models can provide useful models of human genetic diseasessuch as Alzheimer's Disease.

Canadian Patent application 2096911 describes a nucleic acid coding foramyloid precursor protein—cleaving protease, which is associated withAlzheimer's Disease and Down's syndrome. The genetic information may beused to diagnose Alzheimer's disease. The genetic information wasisolated from chromosome 19. Canadian patent application 2071105,describes detection and treatment of inherited or acquired Alzheimer'sdisease by the use of YAC nucleotide sequences. The YACs are identifiedby the numbers 23CB10, 28CA12 and 26FF3.

U.S. Pat. No. 5,297,562, describes detection of Alzheimer's Diseasehaving two or more copies of chromosome 21. Treatment involves methodsfor reducing the proliferation of chromosome 21 trisomy. Canadian PatentApplication 2054302, describes monoclonal antibodies which recognizehuman brain cell nucleus protein encoded by chromosome 21 and are usedto detect changes or expression due to Alzheimer's Disease or Down'sSyndrome. The monoclonal antibody is specific to a protein encoded byhuman chromosome 21 and is linked to large pyramidal cells of humanbrain tissue.

By extensive effort and a unique approach to investigating the AD3region of chromosome 14q, the Alzheimer's related membrane protein(ARMP) gene has been isolated, cloned and sequenced from within the AD3region on chromosome 14q24.3. In addition, direct sequencing of RT-PCRproducts spanning this 3.0 kb cDNA transcript isolated from affectedmembers of at least eight large pedigrees linked to chromosome 14, hasled to the discovery of missence mutations in each of these differentpedigrees. These mutations are absent in normal chromosomes. It has notbeen established that the ARMP gene is causative of familial Alzheimer'sDisease type AD3. In realizing this link, it is understood thatmutations in this gene can be associated with other cognitive,intellectual, or psychological disease such as cerebral hemorrhage,schizophrenia, depression, mental retardation and epilepsy. Thesephenotypes are present in these AD families and these phenotypes havebeen seen in mutations of the APP protein gene. The Amyloid PrecursorProtein (APP) gene is also associated with inherited Alzheimer'sDisease.

The identification of both normal and mutant forms of the ARMP gene andgene products has allowed for the development of screening anddiagnostic tests for ARMP utilizing nucleic acid probes and antibodiesto the gene product. Through interaction with the defective gene productand the pathway in which this gene product is involved, gene therapy,manipulation and delivery are now made possible.

SUMMARY OF THE INVENTION

Various aspects of the invention are summarized as follows. Inaccordance with a first aspect of the invention, a purified mammalianpolynucleotide is provided which codes for Alzheimer's related membraneprotein (ARMP). The polynucleotide has a sequence which is thefunctional equivalent of the DNA sequence of ATCC deposit 97124,deposited Apr. 28, 1995. The mammalian polynucleotide may be in the formof DNA, genomic DNA, cDNA, mRNA and various fragments and portions ofthe gene sequence encoding ARMP. The mammalian DNA is conserved in manyspecies, including human and rodents, example, mice. The mouse sequenceencoding ARMP has greater than 95% homology with the human sequenceencoding the same protein.

Purified human nucleotide sequences which encode mutant ARMP havemutations at nucleotide position i) 685, A→C ii) 737, A→G iii) 986, C→A,iv) 1105, C→G, v) 1478, G→A, vi) 1027, C→T, vii) 1102, C-+T and viii)1422, C→G of Sequence ID No: 1 as well as in the cDNA sequence of afurther human clone of a sequence identified by ID NO: 133.

The nucleotide sequences encoding ARMP have an alternative splice formin the genes open reading frame.

The human cDNA sequence which codes for ARMP has sequence ID No. 1 aswell as sequence SEQ ID NO: 133 as sequenced in another human clone. Themouse sequence which encodes ARMP has SEQ ID NO:3, as well as SEQ ID NO:135 derived from a further clone containing the entire coding region.Various DNA and RNA probes and primers may be made from appropriatepolynucleotide lengths selected from the sequences. Portions of thesequence also encode antigenic determinants of the ARMP.

Suitable expression vectors comprising the nucleotide sequences areprovided along with suitable host cells transfected with such expressionvectors.

In accordance with another aspect of the invention, purified mammalianAlzheimer's related membrane protein is provided. The purified proteinhas an amino acid sequence encoded by polynucleotide sequence asidentified above which for the human is SEQ ID NO:2 and SEQ ID NO: 134(derived from another clone). The mouse amino acid sequence is definedby SEQ ID NO:4 and SEQ ID NO. 136, the latter being translated fromanother clone containing the entire coding region. The purified proteinmay have substitution mutations selected from the group consisting ofpositions identified in SEQ ID NO:2 and Sequence ID NO: 134.

-   -   i) M 146L    -   ii) H 163R    -   iii) A 246E    -   iv) L 286V    -   v) C 410 Y    -   vi) A 260 V    -   vii) A 285 V    -   viii) L 392 V

In accordance with another aspect of the invention, are polyclonalantibodies raised to specific predicted sequences of the ARMP protein.Polypeptides of at least six amino acid residues are provided. Thepolypeptides of six or greater amino acid residues may define antigenicepitopes of the ARMP. Monoclonal antibodies having suitably specificbinding affinity for the antigenic regions of the ARMP are prepared byuse of corresponding hybridoma cell lines. In addition, other polyclonalantibodies may be prepared by inoculation of animals with suitablepeptides or holoprotein which add suitable specific binding affinitiesfor antigenic regions of the ARMP.

In accordance with another aspect of the invention, an isolated DNAmolecule is provided which codes for E5-1 protein. A plasmid includingthis nucleic acid was deposited with the ATCC under the terms of theBudapest Treaty on Jun. 28, 1995 and has been assigned ATCC accessionnumber 97214.

In accordance with another aspect of the invention, purified E5-1protein is provided, having amino acid SEQ ID NO: 138.

In accordance with another aspect of the invention a bioassay isprovided for determining if a subject has a normal or mutant ARMP, wherethe bioassay comprises

providing a biological sample from the subject

conducting a biological assay on the sample to detect a normal or mutantgene sequence coding form ARMP, a normal or mutant ARMP amino acidsequence, or a normal or defective protein function.

In accordance with another aspect of the invention, a process isprovided for producing ARMP comprising culturing one of the abovedescribed transfected host cells under suitable conditions, to producethe ARMP by expressing the DNA sequence. Alternatively, ARMP may beisolated from mammalian cells in which the ARMP is normally expressed.

In accordance with another aspect of the invention, is a therapeuticcomposition comprising ARMP and a pharmaceutically acceptable carrier.

In accordance with another aspect of the invention, a recombinant vectorfor transforming a mammalian tissue cell to express therapeuticallyeffective amounts of ARMP in the cells is provided. The vector isnormally delivered to the cells by a suitable vehicle. Suitable vehiclesinclude vaccinia virus, adenovirus, adeno associated virus, retrovirus,liposome transport, neuraltripic viruses, Herpes simplex virus and othervector systems.

In accordance with another aspect of the invention, a method of treatinga patient deficient in normal ARMP comprising administering to thepatient a therapeutically effective amount of the protein targeted at avariety of patient cells which normally express ARMP. The extent ofadministration of normal ARMP being sufficient to override any effectthe presence of the mutant ARMP may have on the patient. As analternative to protein, suitable ligands and therapeutic agents such assmall molecules and other drug agents may be suitable for drug therapydesigned to replace the protein and defective ARMP, displace mutantARMP, or to suppress its formation.

In accordance with another aspect of the invention an immuno therapy fortreating a patient having Alzheimer's Disease comprises treating thepatient with antibodies specific to the mutant ARMP to reduce biologicallevels or activity of the mutant ARMP in the patient. To facilitate suchamino acid therapy, a vaccine composition may be provided for evoking animmune response in a patient of Alzheimer's disease where thecomposition comprises a mutant ARMP and a pharmaceutically acceptablecarrier with or without a suitable excipient. The antibodies developedspecific to the mutant ARMP could be used to target appropriatelyencapsulated drugs/molecules, specific cellular/tissue sites. Therapiesutilizing specific ligands which bind to normal or wild type ARMP ofeither mutant or wild type and which augments normal function of ARMP inmembranes and/or cells or inhibits the deleterious effect of the mutantprotein are also made possible.

In accordance with another aspect of the invention, a transgenic animalmodel for Alzheimer's Disease which has the mammalian polynucleotidesequence with at least one mutation which when expressed results inmutant ARMP in animal cells and thereby manifests a phenotype. Forexample, the human Prion gene when overexpressed in rodent peripheralnervous system and muscle cells causes a quite different response in theanimal than the human. The animal may be a rodent and is preferably amouse, but may also be other animals including rat, pig, Irosophilamelanogaster, C. elegans (nematode), all of which are used fortransgenic models. Yeast cells can also be used in which the ARMPSequence is expressed from an artificial vector.

In accordance with another aspect of the invention, a transgenic mousemodel for Alzheimer's Disease has the mouse gene encoding ARMP human ormurine homologues mutated to manifest the symptoms. The transgenic mousemay exhibit symptoms of cognitive memory or behavioral disturbances. Inaddition or alternatively, the symptoms may appear as another cellulartissue disorder such as in mouse liver, kidney, spleen or bone marrow orother organs in which the ARMP gene is normally expressed.

In accordance with another aspect of the invention, the protein can beused as a starting point for rationale drug design to provide ligands,therapeutic drugs or other types of small chemical molecules.

BRIEF DESCRIPTION OF THE DRAWINGS

Various aspects of the invention are described hereinafter with respectto the drawings wherein:

FIG. 1 a. Genomic physical and transcriptional map of the AD3 region ofchromosome 14. Genetic map inter-marker genetic distances averaged formale and female meiosis are indicated in centiMorgans.

FIG. 1 b. Is the constructed physical contig map of overlapping genomicDNA fragments cloned into YACs spanning a FAD locus on chromosome 14q.

FIG. 1 c. Regions of interest within the constructed physical contigmap.

FIG. 1 d. Transcriptional map illustrating physical locations of the 19independent longer cDNA clones.

FIG. 2 a. Automated florescent chromatograms representing the change innucleic acids which direct (by the codon) the amino acid sequence of thegene; Met 146 Leu.

FIG. 2 b. Automated florescent chromatograms representing the change innucleic acids which direct (by the codon) the amino acid sequence of thegene; His 163 Arg.

FIG. 2 c. Automated florescent chromatograms representing the change innucleic acids which direct (by the codon) the amino acid sequence of thegene; Ala 246 Glu.

FIG. 2 d. Automated florescent chromatograms representing the change innucleic acids which direct (by the codon) the amino acid sequence of thegene; Leu 286 Val.

FIG. 2 e. Automated florescent chromatograms representing the change innucleic acids which direct (by the codon) the amino acid sequence of thegene; Cys 410 Tyr.

FIG. 3 a. Hydropathy plot of the putative ARMP protein.

FIG. 3 b. A model for the structural organization of the putative ARMPprotein. Roman numerals depict the transmembrane domains. Putativeglycosylation sites are indicated as asterisks and most of thephosphorylation sites are located on the same membrane face as the twoacidic hydrophillic loops. The MAP kinase site is present at residue 115and the PKC site at residue 114. FAD mutation sites are indicated byhorizontal arrows.

FIG. 4 shows the predicted structure of the E5-1 protein.

DETAILED DESCRIPTION OF PREFERRED EMBODIMENTS

In order to facilitate review of the various embodiments of theinvention and an understanding of various elements and constituents usedin making the invention and using same, the following definition ofterms used in the invention description is as follows:

Alzheimer Related Membrane Protein gene (ARMP gene)—the chromosome 14gene which when mutated is associated with familial Alzheimer's Diseaseand/or other inheritable disease phenotypes (e.g., cerebral hemorrhage,mental retardation, schizophrenia, psychosis, and depression). Thisdefinition is understood to include the various sequence polymorphismsthat exist, wherein nucleotide substitutions in the gene sequence do notaffect the essential function of the gene product, as well as functionalequivalents of the nucleotide sequences of SEQ ID NO: 1, SEQ ID NO: 133,SEQ ID NO:3 and SEQ ID NO: 135. This term primarily relates to anisolated coding sequence, but can include some or all of the flankingregulatory elements and/or introns. The term ARMP gene includes the genein other species analogous to the human gene which when mutated isassociated with Alzheimer's Disease.

Alzheimer Related Membrane Protein (ARMP)— the protein encoded by theARMP gene. The preferred source of protein is the mammalian protein asisolated from humans or animals. Alternatively, functionally equivalentproteins may exist in plants, insects and invertebrates (such as C.elegans). The protein may be produced by recombinant organisms, orchemically or enzymatically synthesized. This definition is understoodto include functional variants such as the various polymorphic forms ofthe protein wherein amino acid substitutions or deletions within theamino acid sequence do not affect the essential functioning of theprotein, or its structure. It also includes functional fragments ofARMP.

Mutant ARMP gene—the ARMP gene containing one or more mutations whichlead to Alzheimer's Disease and/or other inheritable disease phenotypes(e.g., cerebral hemorrhage, mental retardation, schizophrenia,psychosis, and depression). This definition is understood to include thevarious mutations that exist, wherein nucleotide substitutions in thegene sequence affect the essential function of the gene product, as wellas mutations of functional equivalents of the nucleotide sequences ofSEQ ID NO: 1, SEQ ID NO: 133, SEQ ID NO:3 and SEQ ID NO: 135 (thecorresponding amino acid sequences). This term primarily relates to anisolated coding sequence, but also can include some or all of theflanking regulatory elements and/or introns.

Mutant ARMP—a mammalian protein that is highly analogous to ARMP interms of primary structure, but wherein one or more amino acid deletionsand/or substitutions result in impairment of its essential function, sothat mammals, especially humans, whose ARMP producing cells expressmutant ARMP rather than the normal ARMP, demonstrate the symptoms ofAlzheimer's Disease and/or other relevant inheritable phenotypes (e.g.cerebral hemorrhage, mental retardation, schizophrenia, psychosis, anddepression).

mARMP gene—mouse gene analogous to the human ARMP gene. Functionalequivalent as used in describing gene sequences and amino acid sequencesmeans that a recited sequence need not be identical to the definitivesequence of the Sequence ID Nos but need only provide a sequence whichfunctions biologically and/or chemically the equivalent of thedefinitive sequence. Hence sequences which correspond to a definitivesequence may also be considered as functionally equivalent sequence.

mARMP—mouse Alzheimer related membrane protein, analogous to the humanARMP, encoded by the mARMP gene. This definition is understood toinclude the various polymorphic forms of the protein wherein amino acidsubstitutions or deletions of the sequence does not affect the essentialfunctioning of the protein, or its structure.

Mutant mARMP—a mouse protein which is highly analogous to mARMP in termsof primary structure, but wherein one or more amino acid deletionsand/or substitutions result in impairment of its essential function, sothat mice, whose mARMP producing cells express mutant mARMP rather thanthe normal mARMP demonstrate the symptoms of Alzheimer's Disease and/orother relevant inheritable phenotypes, or other phenotypes andbehaviours as manifested in mice.

ARMP carrier—a mammal in apparent good health whose chromosomes containa mutant ARMP gene that may be transmitted to the offspring and who willdevelop Alzheimer's Disease in mid to late adult life.

Missense mutation—A mutation of nucleic acid sequence which alters acodon to that of another amino acid, causing an altered translationproduct to be made.

Pedigree—In human genetics, a diagram showing the ancestralrelationships and transmission of genetic traits over severalgenerations in a family.

E5-1 gene—the chromosome 1 gene which shows homology to the ARMP geneand which when mutated is associated with familial Alzheimer's Diseaseand/or other inheritable disease phenotypes. This definition isunderstood to include the various sequence polymorphisms that exist,wherein nucleotide substitutions in the gene sequence do not affect theessential function of the gene product, as well as functionalequivalents of the nucleotide SEQ ID NO:137. This term also includes thegene in other species analogous to the human gene described herein.

E5-1 protein—the protein encoded by the E5-1 gene. This term includesthe protein of SEQ ID NO: 138 and also functional variants such as thevarious polymorphic and splice variant forms of the protein whereinamino acid substitutions or deletions within the amino acid sequence donot affect the essential functioning of the protein. The term alsoincludes functional fragments of the protein.

Mutant E5-1 gene—the E5-1 gene containing one or more mutations whichlead to Alzheimer's Disease. This term is understood to include thevarious mutations that exist, wherein nucleotide substitutions in thegene sequence affect the essential function of the gene product.

Mutant E5-1, protein—a protein analogous to E5-1 protein but wherein oneor more amino acid deletions and/or substitutions result in impairmentof its essential function such that mammals, especially humans, whoseE5-1 producing cells express mutant E5-1 protein demonstrate thesymptoms of Alzheimer's disease.

Linkage analysis—Analysis of co-segregation of a disease trait ordisease gene with polymorphic genetic markers of defined chromosomallocation.

-   -   hARMP gene—human ARMP gene.    -   ORF—pen reading frame.    -   PCR—Polymerase chain reaction.    -   contig—continuous cloned regions.    -   YAC—yeast artificial chromosome.    -   RT-PCR—reverse transcription polymerase chain reaction.    -   SSR—Simple sequence repeat polymorphism.

The present invention is concerned with the identification andsequencing of the mammalian ARMP gene in order to gain insight into thecause and etiology of familial Alzheimer's Disease. From thisinformation, screening methods and therapies for the diagnosis andtreatment of the disease can be developed. The gene has been identified,cDNA isolated and cloned, its transcripts and gene products identifiedand sequenced.

During such identification of the gene, considerable sequenceinformation has also been developed on intron information in the ARMPgene, flanking untranslated information and signal information andinformation involving neighbouring genes in the AD3 chromosome region.Direct sequencing of overlapping RT-PCR products spanning the human geneisolated from affected members of large pedigrees linked to chromosome14 has led to the discovery of missense mutation which co-segregate withthe disease.

Although it is generally understood that Alzheimer's Disease is aneurological disorder, most likely in the brain, expression of ARMP hasbeen found in varieties of human tissue such as heart, brain, placenta,lung, liver, skeletal muscle, kidney and pancreas. Although this gene isexpressed widely, the clinically apparent phenotype exists in brainalthough it is conceivable that biochemical phenotypes may exist inthese other tissues. As with other genetic diseases such as Huntington'sDisease and APP-Alzheimer's, the clinical disease manifestation mayreflect different biochemistries of different cell types and tissues(which stem from genetics and the protein). Such findings suggest thatAD may not be solely a neurological disorder but may also be a systemicdisorder, hence requiring alternative therapeutic strategies which maybe targeted to other tissues or organs or generally in addition orseparately from neuronal or brain tissues.

The ARMP mutations identified have been related to Alzheimer's Diseasepathology. With the identification of sequencing of the gene and thegene product, probes and antibodies raised to the gene product can beused in a variety of hybridization and immunological assays to screenfor and detect the presence of either a normal or mutated gene or geneproduct.

Patient therapy through removal or blocking of the mutant gene product,as well as supplementation with the normal gene product byamplification, by genetic and recombinant techniques or by immunotherapycan now be achieved. Correction or modification of the defective geneproduct by protein treatment immunotherapy (using antibodies to thedefective protein) or knock-out of the mutated gene is now alsopossible. Familial Alzheimer's Disease could also be controlled by genetherapy in which the gene defect is corrected in situ or by the use ofrecombinant or other vehicles to deliver a DNA sequence capable ofexpressing the normal gene product, or a deliberately mutated version ofthe gene product whose effect counter balances the deleteriousconsequences of the disease mutation to the affected cells of thepatient.

The present invention is also concerned with the identification andsequencing of a second gene, the E5-1 gene on chromosome 1, which isassociated with familial Alzheimer's Disease.

Disease mechanism insights and therapies analogous to those describedabove in relation to the ARMP gene will be available as a result of theidentification and isolation of the E5-1 gene.

Isolating the Human ARMP Gene

Genetic Mapping of the AD3 Locus.

After the initial regional mapping of the AD3 gene locus to 14q24.3 nearthe anonymous microsatellite markers D14S43 and D14S53 (Schellenberg, GD et al., 1992; St. George-Hyslop, P et al., 1992; Van Broeckhoven, C etal., 1992), twenty one pedigrees were used to segregate AD as a putativeautosomal dominant trait (St. George-Hyslop P et al., 1992) and toinvestigate the segregation of 18 additional genetic markers from the14q24.3 region which had been organized into a high density geneticlinkage map (FIG. 1 b) (Weissenbach et al., 1992; Gyapay et al., 1994).Pairwise maximum likelihood analyses previously published confirmedsubstantial cumulative evidence for linkage between FAD and all of thesemarkers (Table 1). However, much of the genetic data supporting linkageto these markers were derived from six large early onset pedigrees FAD1(Nee et al., 1983) FAD2 (Frommelt et al., 1991), FAD3 (Goudsmit et al.,1981; Pollen, 1993), FAD4 (Foncin et al., 1985) TOR1.1 (Bergamini, 1991)and 603 (Pericak-Vance et al., 1988) each of which provide at lease oneanonymous genetic marker from 14q24.3 (St. George-Hyslop, P. et al.,1992).

In order to more precisely define the location of the AD3 gene relativeto the known locations of the genetic markers from 14q24.3,recombinational landmarks were sought by direct inspection of the rawhaplotype data only from genotyped affected members of the six pedigreesshowing definitive linkage to chromosome 14. This selective strategy inthis particular instance necessarily discards data from thereconstructed genotypes of deceased affected members as well as fromelderly asymptomatic members of large pedigrees, and takes no account ofthe smaller pedigrees of uncertain linkage status. However, thisstrategy is very sound because it also avoids the acquisition ofpotentially misleading genotype data acquired either through errors inthe reconstructed genotypes of deceased affected members arising fromnon-paternity or sampling errors or from the inclusion of unlinkedpedigrees.

Upon inspection of the haplotype data for affected subjects, members ofthe six large pedigrees whose genotypes were directly determinedrevealed obligate recombinants at D14S48 and D14S53, and at D14S258 andD14S63. The single recombinant at D14S53, which depicts a telomericboundary for the FAD region, occurred in the same AD affected subject ofthe FAD1 pedigree who had previously been found to be recombinant atseveral other markers located telomeric to D14S53 including D14S48 (St.George-Hyslop, P et al., 1992). Conversely, the single recombinant atD14S258, which marks a centromeric boundary of the FAD region, occurredin an affected member of the FAD3 pedigree who was also recombinant atseveral other markers centromeric to D14S258 including D14S63. Bothrecombinant subjects had unequivocal evidence of Alzheimer's Diseaseconfirmed though standard clinical tests for the illness in otheraffected members of their families, and the genotypes of bothrecombinant subjects was informative and co-segregating at multiple lociwithin the interval centromeric to D14S53 and telomeric to D14S258.

When the haplotype analyses were enlarged to include the reconstructedgenotypes of deceased affected members of the six large pedigrees aswell as data from the remaining fifteen pedigrees with probabilities forlinkage of less than 0.95, several additional recombinants were detectedat one or more marker loci within the interval between D14S53 andD14S258. Thus, one additional recombinant was detected in thereconstructed genotype of a deceased affected member of each of three ofthe larger FAD pedigrees (FAD1, FAD2 and other related families), andeight additional recombinants were detected in affected members of fivesmaller FAD pedigrees. However, while some of these recombinants mighthave correctly placed the AD3 gene within a more defined target region,we were forced to regarded these potentially closer “internalrecombinants” as unreliable not only of the reasons discussed earlier,but also because they provided mutually inconsistent locations for theAD3 gene within the D14S5-D14S258 interval.

Construction of a Physical Contig Spanning the AD3 Region.

As an initial step toward cloning the AD3 gene a contig of overlappinggenomic DNA fragments cloned into yeast artificial chromosome vectors,phage artificial chromosome vectors and cosmid vectors was constructed(FIG. 1 b). FISH mapping studies using cosmids derived from the YACclones 932c7 and 964f5 suggested that the interval most likely to carrythe AD3 gene was at least five megabases in size. Because the large sizeof this minimal co-segregating region would make positional cloningstrategies intactable, additional genetic pointers were sought whichfocused the search for the AD3 gene to one or more subregions within theinterval flanked by D14S53 and D14S258. Haplotype analyses at themarkers between D14S53 and D14S258 failed to detect statisticallysignificant evidence for linkage disequilibrium and/or allelicassociation between the FAD trait and alleles at any of these markers,irrespective of whether the analyses were restricted to those pedigreeswith early onset forms of FAD, or were generalized to include allpedigrees. This result was not unexpected given the diverse ethnicorigins of our pedigrees. However, when pedigrees of similar ethnicdescent were collated, direct inspection of the haplotypes observed onthe disease bearing chromosomes segregating in different pedigrees ofsimilar ethnic origin revealed two clusters of marker loci (Table 2).The first of these clusters located centromeric to D14S77 (D14S786,D14S277 and D14S268) and spanned the 0.95 Mb physical interval containedin YAC78842 (depicted as region B in FIG. 1 c). The second cluster waslocated telomeric to D14S77 (D14S43, D14S273, and D14S76) and spannedthe −1 Mb physical interval included within the overlapping YAC clones964c2, 74163, 797d11 and part of 854f5 (depicted as region A in FIG. 1c). Identical alleles were observed in at least two pedigrees from thesame ethnic origin (Table 2). As part of the strategy, it was reasonedthat the presence of shared alleles at one of these groups of physicallyclustered marker loci might reflect the co-inheritance of a smallphysical region surrounding the ARMP gene on the original founderchromosome in each ethnic population. Significantly, each of the sharedextended haplotypes were rare in normal Caucasian populations and allelesharing was not observed at other groups of markers spanning similargenetic intervals elsewhere on chromosome 14q24.3.

Transcription Mapping and Preliminary Analysis of Candidate Genes

To isolate expressed sequences encoded within both critical intervals, adirect selection strategy was used in involving immobilized, cloned,human genomic DNA as the hybridization target to recover transcribedsequences from primary complementary DNA pools derived from human brainmRNA (Rommens et al., 1993). Approximately 900 putative cDNA fragmentsof size 100 to 600 base pairs were recovered from regions A and B inFIG. 1 c. These fragments were hybridized to Southern blots containinggenomic DNAs from each of the overlapping YAC clones and genomic DNAsfrom humans and other mammals. This identified a subset of 151 cloneswhich showed evidence for evolutionary conservation and/or for a complexstructure which suggested that they were derived from spliced mRNA. Theclones within this subset were collated on the basis of physical maplocation, cross-hyridization and nucleotide sequence, and were used toscreen conventional human brain cDNA libraries for longer cDNAs. Atleast 19 independent cDNA clones over 1 kb in length were isolated andthen aligned into a partial transcription map of the AD3 region (FIG. 1d). Only three of these transcripts corresponded to known characterizedgenes (cFOS, dihydrolipoamide succinyl transferase and latenttransforming growth factor binding protein 2).

Recovery of Potential Candidate Genes

Each of the open reading frame portions of the candidate genes wererecovered by RT-PCR from mRNA isolated from post-mortem brain tissue ofnormal control subjects and from either post-mortem brain tissue orcultured fibroblast cell lines of affected members of six pedigreesdefinitively linked to chromosome 14. The RT-PCR products were thenscreened for mutations using chemical cleavage and restrictionendonuclease fingerprinting single-strand sequence conformationalpolymorphism methods (Saleeba and Cotton, 1993; Liu and Sommer, 1995),and by direct nucleotide sequencing. With one exception, all of thegenes examined, although of interest, were not unique to affectedsubjects, and did not co-segregate with the disease. The singleexception was the candidate gene represented by clone S182 whichcontained a series of nucleotide changes not observed in normalsubjects, but which altered the predicted amino acid sequence inaffected subjects. Although nucleotide sequence differences were alsoobserved in some of the other genes, most were in the 3′ untranslatedregions and none were unique to Ad—affected subjects.

The remaining sequences, a subset of which are mapped in FIG. 1 btogether with additional putative transcriptional sequences notidentified in FIG. 1 c, are identified in the sequence listings as 14through 43. The SEQ ID NOS: 14 to 43 represent neighbouring genes orfragments of neighbouring genes adjacent the hARMP gene or possiblyadditional coding fragments arising from alternative splicing of thehARMP. SEQ ID NOS:44-126 and SEQ ID NOS: 150-160 represent neighboringgenomic fragments containing both exon and intron information. Suchsequences are useful for creating primers, for creating diagnostictests, creating altered regulatory sequences and use of adjacent genomicsequences to create better animal models.

Characterization of the hARMP Gene

Hybridization of the S182 clone to northern blots identified atranscript expressed widely in many areas of brain and peripheraltissues as a major 3.0 kb transcript and a minor transcript of 7.0 kb.Although the identity of the 7.0 kb transcript is unclear, twoobservations suggest that the 3.0 kb transcript represents an activeproduct of the gene. Hybridization of the S182 clone to northern blotscontaining mRNA from a variety of murine tissues, including brain,identifies only a single transcript identical in size to the 3.0 kbhuman transcript. All of the longer cDNA clones recovered to date(2.6-2.8 kb), which include both 5′ and 3′ UTRs and which account forthe 3.0 kb band on the northern blot, have mapped exclusively to thesame physical region of chromosome 14. From these experiments the 7.0 kbtranscript could represent either a rare alternatively spliced orpolyadenylated isoform of the ˜3.0 transcript or could represent anothergene with homology to S182

The nucleotide sequence of the major transcript was determined from theconsensus of eleven independent longer cDNA clones and from 3independent clones recovered by standard 5′ rapid amplification of cDNAends and bears no significant homology to other human genes. The cDNA ofthe sequenced transcript is provided in SEQ ID NO: 1 and the predictedamino acid sequence is provided in SEQ ID NO:2. The cDNA sequence ofanother sequenced human clone is also provided as SEQ ID NO: 133 and itspredicted amino acid sequence is provided in SEQ ID NO:134.

Analysis of the 5′ end of multiple cDNA clones and RT-PCR products aswell as corresponding genomic clones indicates that the 5′ UTR iscontained within at least two exons and that transcription either beginsfrom two different start sites and/or that one of the early 5′untranslated exons is alternatively spliced (Table 6). The longestpredicted open reading frame contains 467 amino acids with a smallalternatively spliced exon of 4 aminc acids at 25 codons from theputative start codon (Table 3). This putative start codon is the firstin phase ATG located 63 bp downstream of a TGA stop codon and lacks aclassical Kozak consensus sequences around the first two in-phase ATGsequences (Rogaer et al., in preparation). Like other genes lackingclassical ‘strong’ start codons, the putative 5′ UTR of the humantranscripts are rich in GC.

Comparison of the nucleic acid and predicted amino acid sequences withavailable databases using the BLAST alignment paradigms revealed modestamino acid similarity with the C. elegans sperm integral membraneprotein SPE-4 (p=1.5e²⁵, 24-37% identity over three groups of at leastfifty residues) and weaker similarity to portions of several othermembrane spanning proteins including mammalian chromogranin A and alphasubunit of mammalian voltage dependent calcium channels (Altschul etal., 1990). This clearly established that they are not the same gene.The amino-acid sequence similarities across putative transmembranedomains may occasionally yield alignment that simply arises from thelimited number of hydrophobic amino acids, but there is also extendedsequence alignment between S182 protein and SPE-4 at severalhydrophillic domains. Both the putative S182 protein and SPE-4 arepredicted to be of comparable size (467 and 465 residues, respectively)and to contain at least seven transmembrane domains with a large acidicdomain preceding the final predicted transmembrane domains with a largeacidic domain preceding the final predicted transmembrane domain. TheS182 protein does have a longer predicted hydrophillic region at the Nterminus.

Further investigation of the hARMP has revealed a host of sequencefragments which form the hARMP gene and include intron sequenceinformation, 5′ end untranslated sequence information and 3′ enduntranslated sequence information (Table 6).

Such sequence fragments are identified in Sequence ID Nos. 6 to 13.

Mutations in the S182 Transcript

Direct sequencing of overlapping RT-PCR products spanning the 3.0 kbS182 transcript isolated from affected members of the six largepedigrees linked to chromosome 14 led to the discovery of eight missensemutations in each of the six pedigrees (Table 7, FIG. 2). Each of thesemutations co-segregated with the disease in the respective pedigrees[FIGS. 3( a)(13)(c)(d)(e)], and were absent from 142 unrelatedneurologically normal subjects drawn from the same ethnic origins as theFAD pedigrees (284 unrelated chromosomes).

The location of the gene within the physical interval segregating withAD3 trait, the presence of eight different missense mutations whichco-segregate with the disease train in six pedigrees definitively linkedto chromosome 14, and the absence of these mutations in 284 independentnormal chromosomes cumulatively confirms that the hARMP gene is the AD3locus. Further biologic support for this hypothesis arises from the factthat the residues mutated in FAD kindreds are conserved in evolution(Table 3) and occur in domains of the protein which are also highlyconserved, and from the fact that the S182 gene product is expressed athigh levels in most regions of the brain including the most severelyaffected with AD.

The DNA sequence for the hARMP gene as cloned has been incorporated intoa plasmid Bluescript. This stable vector has been deposited at ATCCunder accession number 97124 on Apr. 28, 1995.

Several mutations in the hARMP gene have been identified which cause asevere type of familial Alzheimer's Disease. One, or a combination ofthese mutations may be responsible for this form of Alzheimer's Diseaseas well as several other neurological disorders. The mutations may beany form of nucleotide sequence alteration or substitution. Specificdisease causing mutations in the form of nucleotide and/or amino acidsubstitutions have been located, although we anticipate additionalmutations will be found in other families. Each of these nucleotidesubstitutions occurred within the putative ORF of the S182 transcript,and would be predicted to change the encoded amino acid at the followingpositions, numbering from the first putative initiation codon. Themutations are listed in respect of their nucleotide locations in SEQ IDNO: 1 and SEQ ID NO: 133 (an additional human clone) and amino acidlocations in SEQ ID NO:2 and SEQ ID NO: 134 (the additional humanclone).

i) 685, A→C Met 146 Leu ii) 737, A→G His 163 Arg iii) 986, C→A Ala 246Glu iv) 1105, C→G Leu 286 Val v) 1478, G→A Cys 410 Tyr vi) 1027, C→T Ala260 Val vii) 1102, C→T Ala 285 Val viii) 1422, C→G Leu 392 Val

The Met146Leu, Ala246Glu and Cys410Tyr mutations have not been detectedin the genomic DNA of affected members of the eight remaining smallearly onset autosomal dominant FAD pedigrees or six additional familiesin our collection which express late FAD onset. We predict that suchmutations would not commonly occur in late onset FAD which has beenexcluded by genetic linkage studies from the more aggressive form of ADlinked to chromosome 14q24.3 (St. George-Hyslop, Pet al., 1992;Schellenberg et al., 1993). The His163Arg mutation has been found in thegenomic DNA of affected members of one additional FAD pedigree for whichpositive but significant statistical evidence for linkage to 14 becomesestablished. Age of onset of affected members was consistent withaffected individuals from families linked to chromosome 14.

Mutations Ala260Val, Ala285Val, and Leu392Val all occur within theacidic hydrophilic loop between putative transmembrane 6 (TM6) andtransmembrane (TM7) (FIG. 6). Two of the mutations (A260V; A285V) andthe L286V mutation are also located in the alternative spliced domain.

All eight of the mutations can be assayed by a variety of strategies(direct nucleotide sequencing, allele specific oligos, ligationpolymerase chain reaction, SSCP, RFLPs etc.) using RT-PCR productsrepresenting the mature mRNA/cDNA sequence or genomic DNA. Allelespecific oligos were chosen for assaying the mutations. For the A260Vand the A285V mutations, genomic DNA carrying the exon was amplifiedusing the same PCR primers and methods as for the L286V mutation. PCRproducts were then denatured and slot blotted to duplicate nylonmembranes using the slot blot protocol described for the C410T mutation.

Of all of the nucleotide substitutions co-segregated with the disease intheir respective pedigrees, none were seen in asymptomatic familymembers aged more than two standard deviations beyond the mean age ofonset, and none were present on 284 chromosomes from unrelatedneurologically normal subjects drawn from comparable ethnic origins.

Identification of an Alternative Splice Form of the ARMP Gene Product

During sequencing studies of RT-PCR products for the ARMP gene recoveredfrom a variety of tissues, it was discovered that some peripheraltissues (principally white blood cells) demonstrated two alternativesplice forms of the ARMP gene. One form is identical to the (putatively467 amino acid) isoform constitutatively expressed in all brain regions.The alternative splice form results from the exclusion of the segment ofthe cDNA between base pairs 1018 and 1116 inclusive, and results in atruncated isoform of the ARMP protein wherein the hydrophobic part ofthe hydrophilic acidically—charged loop immediately C-terminal to TM6 isremoved. This alternatively spliced isoform therefore is characterizedby preservation of the sequence N-terminal to and including the tyrosineat position 256, changing of the aspartate at 257 to alanine, andsplicing on to the C-terminal part of the protein from and includingtyrosine 291. Such splicing differences are often associated withimportant functional domains of the proteins. This argues that thishydrophilic loop (and consequently the N-terminal hydrophilic loop withsimilar amino acid charge) is/are active functional domains of the ARMPproduct and thus sites for therapeutic targeting.

ARMP Protein

With respect to DNA SEQ ID NO. 1 and DNA SEQ ID NO: 133, analysis of thesequence of overlapping cDNA clones predicted an ORF protein of 467amino acids when read from the first in phase ATG start codon and amolecular mass of approximately 52.6 kDa as later described, due toeither polymorphisms in the protein or alternate splicing of thetranscript, the molecular weight of the protein can vary due to possiblesubstitutions or deletions of amino acids.

The analysis of predicted amino acid sequence using the Hopp and Woodsalgorithm suggested that the protein product is a multispanning integralmembrane protein such as a receptor, a channel protein, or a structuralmembrane protein. The absence of recognizable signal peptide and thepaucity of glycoslyation sites are noteworthy, and the hydropathyprofile suggests that the protein is less likely to be a soluble proteinwith a highly compact three-dimensional structure.

The protein may be a cellular protein with a highly compact threedimensional structure in which respect is may be similar to APOE whichis also related to Alzheimer's Disease. In light of this putativefunctional role, it is proposed that this protein be labeled as theAlzheimer Related Membrane Protein (ARMP). The protein also contains anumber of potential phosphorylation sites, one of which is the consensussite for MAPkinase which is also involved in the hyperphosphorlyation oftau during the normal conversion of normal tau to neurofibrillarytangles. This consensus sequence may provide a common putative pathwaylinking this protein and other known biochemical aspects of Alzheimer'sDisease and would represent a likely therapeutic target. Review of theprotein structure reveals two sequence YTPF (residues 115-119) BEQ IDNO: 161 and STPE (residues 353-356) SEQ ID NO: 162 which represent the5/T-P motif which is the MAP kinase consensus sequence. Several otherphosphorylation sites exist with consensus sequences for Protein KinaseC activity. Because protein kinase C activity is associated withdifferences in the metabolism of APP which are relevant to Alzheimer'sDisease, these sites on the ARMP protein and homologues are sites fortherapeutic targeting.

The N-terminal is characterized by a highly hydrophilic acid chargeddomain with several potential phosphorylation domains, followedsequentially by a hydrophobic membrane spanning domain of 19 residues; acharged hydrophilic loop, then five additional hydrophobic membranespanning domains interspersed with short (5-20 residue) hydrophilicdomains; an additional larger acidic hydrophilic charged loop, and thenat least one and possibly two other hydrophobic potentially membranespanning domains culminating in a polar domain at the C-terminus (Table4 and FIG. 6B). The presence of seven membrane spanning domains ischaracteristic of several classes of G—coupled receptor proteins but isalso observed with other proteins including channel proteins.

Comparison of the nucleic acid and predicted amino acid sequences withavailable databases using the BLAST alignment paradigms revealed aminoacid similarity with the C. elegans sperm integral membrane proteinSPE-4 and a similarity to several other membrane spanning proteinsincluding mammalian chromogranin A and the a-subunit of mammalianvoltage dependent calcium channels.

The similarity between the putative products of the SPE-4 and ARMP genesimplies that they may have similar activities. The SPE-4 protein of C.elegans appears to be involved in the formation and stabilization of thefibrous body-membrane organelle (FMBO) complex during spermatogenesis.The FBMO is a specialized Golgi-Derived organelle, consisting of amembrane bound vesicle attached to and partly surrounding a complex ofparallel protein fibers and may be involved in the transport and storageof soluble and membrane-bound polypeptides. Mutations in SPE-4 disruptthe FBMO complexes and arrest spermatogenesis. Therefore the physiologicfunction of SPE-4 may be either to stabilize interactions betweenintegral membrane budding and fusion events, or to stabilizeinteractions between the membrane and fibrillary proteins during theintracellular transport of the FBMO complex during spermatogenesis.Comparable functions could be envisaged for the ARMP. The ARMP could beinvolved either in the docking of other membrane-bound proteins such asPPP, or the axonal transport and fusion budding of membrane-boundvesicles during protein transport such as in the golgi apparatus orendosome-lysosome system. If correct, then mutations might be expectedto result in aberrant transport and processing of PAPP and/or abnormalinteractions with cytoskeletal proteins such as themicrotubule-associated protein Tau. Abnormalities in the intracellularand in the extracellular disposition of both PAPP and Tau are in fact anintegral part of the neuropathologic features of Alzheimer's Disease.Although the location of the ARMP mutations in highly conserved residueswithin conserved domains of the putative proteins suggests that they arepathogenic, at least three of these mutations are conservative which iscommensurate with the onset of disease in adult life. Because none ofthe mutations observed so far are deletions or nonsense mutations thatwould be expected to cause a loss of function, we cannot predict whetherthese mutations will have a dominant gain-of-function effect and promoteaberrant processing of βAPP or a dominant loss-of-function effectcausing arrest of normal βAPP processing.

An alternative possibility is that the ARMP gene product may represent areceptor or channel protein. Mutations of such proteins have beencausally related to several other dominant neurologic disorders in bothvertebrate (e.g., Malignant hyperthermia, hyperkalemic periodicparalysis in humans) and in invertebrate organisms (deg-1(d) mutants inC. elegans). Although the pathology of these other disorders does notresemble that of Alzheimer's Disease there is evidence for functionalabnormalities in ion channels in Alzheimer's Disease. For example,anomalies have been reported in the tetra-ethylammonium-sensitive 113pSpotassium channel and in calcium homeostasis. Perturbations intransmembrane calcium fluxes might be especially relevant in view of theweak homology between S1182 and the α-ID subunit of voltage-dependentcalcium channels and the observations that increases in intracellularcalcium in cultured cells can replicate some of the biochemical featuresof Alzheimer's Disease such as alteration in the phosphorylation ofTau-microtubule associated protein and increased production of Aβpeptides.

As mentioned purified normal ARMP protein is characterized by amolecular weight of 52.6 kDa. The normal ARMP protein, substantiallyfree of other proteins, is encoded by the aforementioned SEQ ID NO:. 1and SEQ ID NO: 133. As will be later discussed, the ARMP protein andfragments thereof may be made by a variety of methods. Purified mutantARMP protein is characterized by FAD—associated phenotype (necroticdeath, apoptic death, granulovascular degeneration, neurofibrillarydegeneration, abnormalities or changes in the metabolism of APP, andCa²⁺, K⁺ and glucose, and mitochondrial function and energy metabolismneurotransmitter metabolism, all of which have been found to be abnormalin human brain, and/or peripheral tissue cells in subjects withAlzheimer's Disease) in a variety of cells. The mutant ARMP, free ofother proteins, is encoded by the mutant DNA sequence.

Description of the E5-1 Gene, a Homologue of the ARMP Gene

A gene, E5-1, with substantial nucleotide and amino acid homology to theARMP gene was identified by using the nucleotide sequence of the cDNAfor ARMP to search data bases using the BLASTN paradigm of Atschul etal., 1990. Three expressed sequence tagged sites (ESTs) identified byaccession numbers T03796, R14600, and R05907 were located which hadsubstantial homology (p<1.0 e⁻¹⁰⁰, greater than 97% identity over atleast 100 contiguous base pairs).

Oligonucleotide primers were produced from these sequences and used togenerate PCR products by reverse transcriptase PCR (RT-PCR). These shortRT-PCR products were partially sequenced to confirm their identity withthe sequences within the data base and were then used as hybridizationprobes to screen full-length cDNA libraries. Several different cDNA'sranging in size from 1 Kb to 2.3 Kb were recovered from a cancer cellcDNA library (CaCo-2) and from a human brain cDNA library (E5-1, G1-1,cc54, cc32).

The nucleotide sequence of these clones confirmed that all werederivatives of the same transcript (designated E5-1). A plasmidincluding this nucleic acid was deposited with the ATCC under the termsof the Budapest Treaty on Jun. 28, 1995 and has been assigned ATCCaccession number 97214.

The gene encoding the E5-1 transcript mapped to human chromosome 1 usinghybrid mapping panels and to two clusters of CEPH Mega YAC clones whichhave been placed upon a physical contig map (YAC clones 750g7, 921d12mapped by FISH to 1q41; and YAC clone 787g12 which also contains an ESTof the leukemia associated phosphoproteins (LAP18) gene which has beenmapped to 1p36.1-p35) (data not shown).

Hybridization of the E5-1 cDNA clones to Northern Blots detected an ˜2.3kilobase mRNA band in many tissues including regions of the brain, aswell as a ˜2.6K.b mRNA band in muscle, cardiac muscle and pancreas (FIG.7).

In skeletal muscle, cardiac muscle and pancreas, the E5-1 gene isexpressed at relatively higher levels than in brain and as two differenttranscripts of ˜2.3 Kb and ˜2.6 Kb. Both of the E5-1 transcripts havesizes clearly distinguishable from that of the 2.7 Kb ARMP transcript,and did not cross-hybridize with ARMP probes at high stringency. ThecDNA sequence of the E5-1 gene is identified as SEQ ID NO.: 137.

The longest ORF within the E5-1 cDNA consensus nucleotide sequencepredicts a polypeptide containing 448 amino acids (numbering from thefirst in-phase ATG codon which was surrounded by a GCC-agg-GCt-ATG-αKozak consensus sequence) (SEQ ID NO.:138) A comparison of the aminoacid sequences of hARMP and E5-1 homologue protein are shown in Table 8.Identical residues are indicated by vertical lines. The locations ofmutations in the E5-1 gene are indicated by downward pointing arrows.The locations of the mutations in the hARMP gene are indicated by upwardpointing arrows. Putative TM domains are in open ended boxes. Thealternatively spliced exons are denoted by superscripted (E5-1) orsubscripted (hARMP) “*”.

BLASTP alignment analyses also detected significant homology with SPE-4of C. elegans (P=3.5e-26; identity=20-63% over five domains of at least22 residues), and weak homologies to brain sodium channels (alpha IIIsubunit) and to the alpha subunit of voltage dependent calcium channelsfrom a variety of species (P=0.02; identities 20-28% over two or moredomains each of at least 35 residues) (Atschul, 1990). These alignmentsare similar to those described above for the ARMP gene. However, themost striking homology to the E5-1 protein was found with the amino acidsequence predicted for ARMP. ARMP and E5-1 proteins share 63% overallamino acid sequence identity, and several domains display virtuallycomplete identity (Table 8). Furthermore, all eight residues mutated inARMP in subjects with AD3 are conserved in the E5-1 protein (Table 8).As would be expected, hydrophobicity analyses suggest that both proteinsalso share a similar structural organization.

The similarity was greatest in several domains of the proteincorresponding to the intervals between transmembrane domain 1 (TM1) andTM6, and from TM7 to the C-terminus of the ARMP gene. The maindifference from ARMP is a difference in the size and amino acid sequenceof the acidically-charged hydrophilic loop in the position equivalent tothe hydrophilic loop between transmembrane domains TM6 and TM7 in theARMP protein and in the sequence of the N-terminal hydrophilic domains.

Thus, both proteins are predicted to possess seven hydrophobic putativetransmembrane domains, and both proteins bear large acidic hydrophilicdomains at the N-terminus and between TMG and TM7 (FIGS. 6 and 8). Afurther similarity arose from analysis of RT-PCR products from brain andmuscle RNA, which revealed that nucleotides 1153-1250 of the E5-1transcript are alternatively spliced. These nucleotides encode aminoacids 263-296, which are located within the TM6-TM7 loop domain of theputative E5-1 protein, and which share 94% sequence identity with thealternatively spliced residues 257-290 in ARMP.

The most noticeable differences between the two predicted amino acidsequences occur in the amino acid sequence in the central portion of theTM6-TM7 hydrophilic loop (residues 304-374 of ARMP; 301-355 of E5-1),and in the N-terminal hydrophilic domain (Table 8). By analogy, thisdomain is also less highly conserved between the murine and human ARMPgenes (identity=47/60 residues), and shows no similarity with theequivalent region of SPE-4.

A splice variant of the E5-1 cDNA sequence identified as SEQ ID NO: 137has also been found in all tissues examined. This splice variant lacksthe tripet GAA at nucleotide positions 1338-1340.

A further variant has been found in one normal individual whose E5-1cDNA had C replacing T at nucleotide position 626, which does not changethe amino acid sequence.

Mutations of the E5-1 Gene Associated with Alzheimer's Disease

The strong similarity between ARMP and the E5-1 gene product raised thepossibility that the E5-1 gene might be the site of disease-causingmutations in some of a small number of early onset AD pedigrees in whichgenetic linkage studies have excluded chromosomes 14, 19 and 21. RT-PCRwas used to isolate cDNAs corresponding to the E5-1 transcript fromlymphoblasts, fibroblasts or post-mortem brain tissue of affectedmembers of eight pedigrees with early onset familial AD (FAD) in whichmutations in the D APP and ARMP gene had previously been excluded bydirect sequencing studies.

Examination of these RT-PCR products detected a heterozygous A→Gsubstitution at nucleotide 1080 in all four affected members of anextended pedigree of Italian origin (Flo10) with early onset,pathologically confirmed FAD (onset=50-70 yrs.). This mutation would bepredicted to cause a Met→Val missense mutation at codon 239 (Table 8).

A second mutation (A→T at nucleotide 787) causing a Asn→Ile substitutionat codon 141 was found in affected members of a group of relatedpedigrees of Volga German ancestry (represented by cell lines AG09369,AG09907, AG09952, and AG09905, Coriell Institute, Camden, N.J.).Significantly, one subject (AG09907) was homozygous for this mutation,an observation compatible with the in-bred nature of these pedigrees.Significantly, this subject did not have a significantly differentclinical picture from those subjects heterozygous for the Arg14Ilemutation. Neither of the E5-1 gene mutations were found in 284 normalCaucasian controls nor were they present in affected members ofpedigrees with the AD3 type of AD.

Both of these mutations would be predicted to cause substitutions ofresidues which are highly conserved within the ARMP/E5-1 gene family.

The finding of a gene whose product is predicted to share substantialamino acid and structural similarities with the ARMP gene productsuggest that these proteins may be functionally related either asindependent proteins with overlapping functions but perhaps withslightly different specific activities, as physically associatedsubunits of a multimeric polypeptide or as independent proteinsperforming consecutive functions in the same pathway.

The observation of two different missense mutations in conserved domainsof the E5-1 protein in subjects with a familial form of AD argues thatthese mutations are, like those in the ARMP gene, causal to AD. Thisconclusion is significant because, while the disease phenotypesassociated with mutations in the ARMP gene (onset 30-50 yrs., duration10 years) are subtly different from that associated with mutations inthe E5-1 gene (onset 40-70 years; duration up to 20 years), the generalsimilarities clearly argue that the biochemical pathway subsumed bymembers of this gene family is central to the genesis of at least earlyonset AD. The subtle differences in disease phenotype may reflect alower level of expression of the E5-1 transcript in the CNS, or mayreflect a different role for the E5-1 gene product.

By analogy to the effects of ARMP mutations, E5-1 when mutated may causeaberrant processing of APP (Amyloid Precursor Protein) into AP peptide,hyperphosphorylation of Tau microtubule associated protein andabnormalities of intracellular calcium homeostasis. Interference withthese anomalous interactions provides a potential therapy for AD.

Functional Domains of the ARMP Protein are Defined by Splicing Sites andSimilarities Within Other Members of a Gene Family

The ARMP protein is a member of a novel class of transmembrane proteinswhich share substantial amino acid homology. The homology is sufficientthat certain nucleotides probes and antibodies raised against one canidentify other members of this gene family. The major difference betweenmembers of this family reside in the amino acid and nucleotide sequencehomologous to the hydrophillic acid loop domain between putativetransmembrane 6 and transmembrane 7 domains of the ARMP gene and geneproduct. This region is alternatively spliced in some non-neuraltissues, and is also the site of several pathogenic disease-causingmutations in the ARMP gene. The variable splicing of this hydrophilicloop, the presence of a high-density of pathogenic mutations within thisloop, and the fact that the amino acid sequences of the loop differsbetween members of the gene family suggest that this loop is animportant functional domain of the protein and may confer somespecificity to the physiologic and pathogenic interactions which theARMP gene product undergoes because the N-terminal hydrophilic domainshares the same acidic charge and same orientation with respect to themembrane, it is very likely that these two domains share functionalityeither in a coordinated (together) or independent fashion (e.g.,different ligands or functional properties). As a result everything saidabout the hydrophilic loop shall apply also to the N-terminalhydrophilic domain.

Knowledge of the specificity of the loop can be used to identify ligandsand functional properties of the ARMP gene product (e.g. sites ofinteractions with APP, cytosolic proteins such as kinases, Tau, and MAP,etc.). Soluble recombinant fusion proteins can be made or the nucleotidesequence coding for amino acids within the loop or parts of the loop canbe expressed in suitable vectors (yeast-2-hybrid, baculovirus, andphage-display systems for instance), and used to identify other proteinswhich interact with ARMP in the pathogenesis of Alzheimer's Disease andother neurological and psychiatric diseases. Therapies can be designatedto modulate these interactions and thus to modulate Alzheimer's Diseaseand the other conditions associated with acquired or inheritedabnormalities of the ARMP gene or its gene products. The potentialefficacy of these therapies can be tested by analyzing the affinity andfunction of these interactions after exposure to the therapeutic agentby standard pharmacokinetic measurements of affinity (Kd and Vmax etc.)using synthetic peptides or recombinant proteins corresponding tofunctional domains of the ARMP gene (or its homologues). An alternatemethod for assaying the effect of any interactions involving functionaldomains such as the hydrophilic loop is to monitor changes in theintracellular trafficking and post-translational modification of theARMP gene by in-situ hybridization, immunohistochemistry, Westernblotting and metabolic pulse-chase labeling studies in the presence ofand in the absence of the therapeutic agents. A third way is to monitorthe effects of “downstream” events including (i) changes in theintracellular metabolism, trafficking and targeting of APP and itsproducts; (ii). changes in second messenger event e.g, cAMP,intracellular Ca kinase activities, etc. Isolation and Purification ofthe ARMP Protein.

The ARMP protein may be isolated and purified by methods selected on thebasis of properties revealed by its sequence. Since the proteinpossesses properties of a membrane-spanning protein, a membrane fractionof cells in which the protein is highly expressed (e.g., central nervoussystem cells or cells from other tissues) would be isolated and theproteins removed by extraction and the proteins solubilized using adetergent.

Purification can be achieved using protein purification procedures suchas chromatography methods (gel-filtration, ion-exchange andimmunoaffinity), by high-performance liquid chromatography (RP-HPLC,ion-exchange HPLC, size-exclusion HPLC, high-performancechromatofocusing and hydrophobic interaction chromatography) or byprecipitation (immunoprecipitation). Polyacrylamide gel electrophoresiscan also be used to isolate the ARMP protein based on its molecularweight, charge properties and hydrophobicity.

Similar procedures to those just mentioned could be used to purify theprotein from cells transfected with vectors containing the ARMP gene(e.g., baculovirus system, yeast expression systems, eukaryoticexpression systems).

Purified protein can be used in further biochemical analyses toestablish secondary and tertiary structure which may aid in the designof pharmaceuticals to interact with the protein, alter protein chargeconfiguration or charge interaction with other proteins, lipid orsaccharide moieties, alter its function in membranes as a transporterchannel or receptor and/or in cells as an enzyme or structural proteinand treat the disease.

The protein can also be purified by creating a fusion protein bylegating the ARMP cDNA sequence to a vector which contains sequence foranother peptide (e.g., GST-glutathionine succinyl transferase). Thefusion protein is expressed and recovered from prokaryotic (e.g.,bacterial or baculovirus) or eukaryotic cells. The fusion protein canthen be purified by affinity chromatography based upon the fusion vectorsequence. The ARMP protein can then be further purified from the fusionprotein by enzymatic cleavage of the fusion protein.

Isolating Mouse ARMP Gene

In order to characterize the physiological significance of the normaland mutant hARMP gene and gene products in a transgenic mouse model itwas necessary to recover a mouse homologue of the hARMP gene. Werecovered a murine homologue for the hARMP gene by screening a mousecDNA library with a labelled human DNA probe and in this mannerrecovered a 2 kb partial transcript (representing the 3′ end of thegene) and several RT-PCR products representing the 5′ end. Sequencing ofthe consensus cDNA transcript of the murine homologue revealedsubstantial amino acid identity. The sequence cDNA is identified in SEQID NO:3 and the predicted amino acid sequence is provided in SEQ IDNO:4. Further sequencing of the mouse cDNA transcript has provided thesequence of the complete coding sequence identified as SEQ ID NO: 135and the predicted amino acid sequence from this sequence is provided inSEQ ID NO:136. More importantly, all of the amino acids that weremutated in the FAD pedigrees were conserved between the murine homologueand the normal human variant (Table 3). This conservation of the ARMPgene as is shown in Table 3, indicates that an orthologous gene existsin the mouse (mARMP), and it is now possible to clone mouse genomiclibraries using human ARMP probes. This will also make it possible toidentify and characterize the ARMP gene in other species. This alsoprovides evidence of animals with various disease states or disorderscurrently known or yet to be elucidated.

Transgenic Mouse Model

The creation of a mouse model for Alzheimer's Disease is important tothe understanding of the disease and for the testing of possibletherapies. Currently no unambiguous viable animal model for Alzheimer'sDisease exists.

There are several ways in which to create an animal model forAlzheimer's Disease. Generation of a specific mutation in the mouse genesuch as the identified hARMP gene mutations is one strategy. Secondly,we could insert a wild type human gene and/or humanize the murine geneby homologous recombination. Thirdly, it is also possible to insert amutant (single or multiple) human gene as genomic or minigene cDNAconstructs using wild type or mutant or artificial promoter elements.Fourthly, knock-out of the endogenous murine genes may be accomplishedby the insertion of artificially modified fragments of the endogenousgene by homologous recombination. The modifications include insertion ofmutant stop codons, the deletion of DNA sequences, or the inclusion ofrecombination elements (lcx p sites) recognized by enzymes such as Crerecombinase.

To inactivate the mARMP gene chemical or x-ray mutagenesis of mousegametes, followed by fertilization, can be applied. Heterozygousoffspring can then be identified by Southern blotting to demonstrateloss of one allele by dosage, or failure to inherit one parental alleleusing RFLP markers.

To create a transgenic mouse a mutant version of hRMP or mARMP can beinserted into a mouse germ line using standard techniques of oocytemicroinjection or transfection or microinjection into stem cells.Alternatively, if it is desired to inactivate or replace the endogenousmARMP gene, homologous recombination using embryonic stem cells may beapplied.

For oocyte injection, one or more copies of the mutant or wild type ARMPgene can be inserted into the pronucleus of a just-fertilized mouseoocyte. This oocyte is then reimplanted into a pseudo-pregnant fostermother. The liveborn mice can then be screened for integrants usinganalysis of tail DNA for the presence of human ARMP gene sequences. Thetransgene can be either a complete genomic sequence injected as a YAC,BAC, PAC or other chromosome DNA fragment, a cDNA with either thenatural promoter or a heterologous promoter, or a minigene containingall of the coding region and other elements found to be necessary foroptimum expression.

Retroviral infection of early embryos can also be done to insert themutant or wild type hARMP. In this method, the mutant or wild type hARMPis inserted into a retroviral vector which is used to directly infectmouse embryos during the early stages of development to generate achimera, some of which will lead to germline transmission. Similarexperiments can be conducted in the cause of mutant proteins, usingmutant murine or other animal ARMP gene sequences.

Homologous recombination using stem cells allows for screening of genetransfer cells to identify the rare homologous recombination events.Once identified, these can be used to generate chimeras by injection ofmouse blastocysts, and a proportion of the resulting mice will showgermline transmission from the recombinant line. This methodology isespecially useful if inactivation of the mARMP gene is desired. Forexample, inactivation of the mARMP gene can be done by designing a DNAfragment which contains sequences from a mARMP exon flanking aselectable marker. Homologous recombination leads to the insertion ofthe marker sequences in the middle of an exon, inactivating the mARMPgene. DNA analysis of individual clones can then be used to recognizethe homologous recombination events.

It is also possible to create mutations in the mouse germline byinjecting oligonucleotides containing the mutation of interest andscreening the resulting cells by PCR.

This embodiment of the invention has the most significant commercialvalue as a mouse model for Alzheimer's Disease. Because of the highpercentage of sequence conservation between human and mouse it iscontemplated that an orthologous gene will exist also in many otherspecies. It is thus contemplated that it will be possible to generateother animal models using similar technology.

Screening and Diagnosis for Alzheimer's Disease General Diagnostic Usesof the ARMP Gene and Gene Product

The ARMP gene and gene products will be useful for diagnosis ofAlzheimer's Disease, presenile and senile dementias, psychiatricdiseases such as schizophrenia, depression, etc., and neurologicdiseases such as stroke and cerebral hemorrhage—all of which are seen toa greater or lesser extent in symptomatic subjects bearing mutations inthe ARMP gene or in the APP gene. Diagnosis of inherited cases of thesediseases can be accomplished by analysis of the nucleotide sequence(including genomic and cDNA sequences included in this patent).Diagnosis can also be achieved by monitoring alterations in theelectrophoretic mobility and by the reaction with specific antibodies tomutant or wild-type ARMP gene products, and by functional assaysdemonstrating altered function of the ARMP gene product. In addition,the ARMP gene and ARMP gene products can be used to search for inheritedanomalies in the gene and/or its products (as well as those of thehomologous gene) and can also be used for diagnosis in the same way asthey can be used for diagnosis of non-genetic cases.

Diagnosis of non-inherited cases can be made by observation ofalterations in the ARMP transcription, translation, andpost-translational modification and processing as well as alterations inthe intracellular and extracellular trafficking of ARMP gene products inthe brain and peripheral cells. Such changes will include alterations inthe amount of ARMP messenger RNA and/or protein, alteration inphosphorylation state, abnormal intracellular location/distribution,abnormal extracellular distribution, etc. Such assays will include:Northern Blots (with ARMP-specific and ARMP non-specific nucleotideprobes which also cross-react with other members of the gene family),and Western blots and enzyme=linked immunosorbent assays (ELISA) (withantibodies raised specifically to: ARMP; to various functional domainsof ARMP; to other members of the homologous gene family; and to variouspost-translational modification states including glycosylated andphosphorylated isoforms). These assays can be performed on peripheraltissues (e.g., blood cells, plasma, cultured or other fibroblasttissues, etc) as well as on biopsies of CNS tissues obtained antimortemor postmortem, and upon cerebrospinal fluid. Such assays might alsoinclude in-situ hybridization and immunohistochemistry (to localizedmessenger RNA and protein to specific subcellular compartments and/orwithin neuropathological structures associated with these diseases suchas neurofibrillary tangles and amyloid plaques).

Screening for Alzheimer's Disease

Screening for Alzheimer's Disease as linked to chromosome 14 may now bereadily carried out because of the knowledge of the mutations in thegene.

People with a high risk for Alzheimer's Disease (present in familypedigree) or, individuals not previously known to be at risk, or peoplein general may be screened routinely using probes to detect the presentof a mutant ARMP gene by a variety of techniques. Genomic DNA used forthe diagnosis may be obtained from body cells, such as those present inthe blood, tissue biopsy, surgical specimen, or autopsy material. TheDNA may be isolated and used directly for detection of a specificsequence or may be PCR amplified prior to analysis. RNA or cDNA may alsobe used. To detect a specific DNA sequence hybridization using specificoligonucleotides, direct DNA sequencing, restriction enzyme digest,RNase protection, chemical cleavage, and ligase-mediated detection areall methods which can be utilized. Oligonucleotides specific to mutantsequences can be chemically synthesized and labelled radioactively withisotopes, or non-radioactively using biotin tags, and hybridized toindividual DNA samples immobilized on membranes or other solid-supportsby dot-blot or transfer from gels after electrophoresis. The presence orabsence of these mutant sequences are then visualized using methods suchas autoradiography, fluorometry, or calorimetric reaction. Examples ofsuitable PCR primers which are useful for example in amplifying portionsof the subject sequence containing the aforementioned mutations are setout in Table 5. This table also sets out the change in enzyme site toprovide a useful diagnostic tool as defined herein.

Direct DNA sequencing reveals sequence differences between normal andmutant ARMP DNA. Cloned DNA segments may be used as probes to detectspecific DNA segments. PCR can be used to enhance the sensitivity ofthis method. PCR is an enzymatic amplification directed bysequence-specific primers, and involves repeated cycles of heatdenaturation of the DNA, annealing of the complementary primers andextension of the annealed primer with a DNA polymerase. This results inan exponential increase of the target DNA.

Other nucleotide sequence amplification techniques may be used, such asligation-mediated PCR, anchored PCR and enzymatic amplification as wouldbe understood by those skilled in the art.

Sequence alterations may also generate fortuitous restriction enzymerecognition sites which are revealed by the use of appropriate enzymedigestion followed by gel-blot hybridization. DNA fragments carrying thesite (normal or mutant) are detected by their increase or reduction insize, or by the increase or decrease of corresponding restrictionfragment numbers. Genomic DNA samples may also be amplified by PCR priorto treatment with the appropriate restriction enzyme and the fragmentsof different sizes are visualized under UV light in the presence ofethidium bromide after gel electrophoresis.

Genetic testing based on DNA sequence differences may be achieved bydetection of alteration in electrophoretic mobility of DNA fragments ingels. Small sequence deletions and insertions can be visualized by highresolution gel electrophoresis. Small deletions may also be detected aschanges in the migration pattern of DNA heteroduplexes in non-denaturinggel electrophoresis. Alternatively, a single base substitution mutationmay be detected based on differential PCR product length in PCR. The PCRproducts of the normal and mutant gene could be differentially detectedin acrylamide gels.

Nuclease protection assays (Si or ligase-mediated) also reveal sequencechanges at specific location.

Alternatively, to confirm or detect a polymorphism restriction mappingchanges ligated PCR, ASO, REF-SSCP chemical cleavage, endonucleasecleavage at mismatch sites and SSCP may be used. Both REF-SSCP and SSCPare mobility shift assays which are based upon the change inconformation due to mutations.

DNA fragments may also be visualized by methods in which the individualDNA samples are not immobilized on membranes. The probe and targetsequences may be in solution or the probe sequence may be immobilized.Autoradiography, radioactive decay, spectrophotometry, and fluorometrymay also be used to identify specific individual genotypes. Finally,mutations can be detected by direct nucleotide sequencing.

According to an embodiment of the invention, the portion of the cDNA orgenomic DNA segment that is informative for a mutation, can be amplifiedusing PCR. For example, the DNA segment immediately surrounding theC410Y mutation acquired from peripheral blood samples from an individualcan be screened using the oligonucleotide primers 885(tggagactggaacacaac) SEQ ID NO: 127 and 893 (gtgtggccagggtagagaact) SEQID NO: 128. This region would then be amplified by PCR, the productsseparated by electrophoresis, and transferred to membrane. Labelledoligonucleotide probes are then hybridized to the DNA fragments andautoradiography performed.

ARMP Expression

As an embodiment of the present invention, ARMP protein may be expressedusing eukaryotic and prokaryotic expression systems. Eukaryoticexpression systems can be used for many studies of the ARMP gene andgene product including determination of proper expression andpost-translational modifications for full biological activity,identifying regulatory elements located in the 5′ region of the ARMPgene and their role in tissue regulation of protein expression,production of large amounts of the normal and mutant protein forisolation and purification, to use cells expressing the ARMP protein asa functional assay system for antibodies generated against the proteinor to test effectiveness of pharmacological agents, or as a component ofa signal transduction system, to study the function of the normalcomplete protein, specific portions of the protein, or of naturallyoccurring and artificially produced mutant proteins.

Eukaryotic and prokaryotic expression systems were generated using twodifferent classes of ARMP nucleotide cDNA sequence inserts. In the firstclass, termed full-length constructs, the entire ARMP cDNA sequence isinserted into the expression plasmid in the correct orientation, andincludes both the natural 5′ UTR and 3′ UTR sequences as well as theentire open reading frame. The open reading frames bear a nucleotidesequence cassette which allows either the wild type open reading frameto be included in the expression system or alternatively, a single or acombination of double mutations can be inserted into the open readingframe. This was accomplished by removing a restriction fragment from thewild type open reading frame using the enzymes Nan I and PflmI andreplacing it with a similar fragment generated by reverse transcriptasePCR which bears the nucleotide sequence encoding either the Met146Leumutation or the Hys163Arg mutation. A second restriction fragment wasremoved from the wild type normal nucleotide sequence for the openreading frame by cleavage with the enzymes PflmI and NcoI and replacedwith restriction fragments bearing either nucleotide sequence encodingthe Ala246Glu mutation, or the Ala260Val mutation or the Ala285Valmutation or the Leu286Val mutation, or the Leu392Val mutation, or theCys410Tyr mutation. Finally, a third variant bearing combinations ofeither the Met146Leu or His163Arg mutations in tandem with the remainingmutations, was made by linking the NarI-PflmI fragment bearing thesemutations and the PflmI-NcoI fragments bearing the remaining mutations.

A second variant of cDNA inserts bearing wild type or mutant cDNAsequences was constructed by removing from the full-length cDNA the 5′UTR and part of the 3′ UTR sequences. The 5′ UTR sequence was replacedwith a synthetic oligonucleotide containing a KpnI restriction site anda Kozak initiation site (oligonucleotide 969:ggtaccgccaccatgacagaggtacctgcac, SEQ ID NO: 139). The 3′ UTR wasreplaced with an oligonucleotide corresponding to position 2566 of thecDNA and bears an artificial EcoRI site (oligonucleotide 970:gaattcactggctgtagaaaaagac, SEQ ID NO: 140). Mutant variants of thisconstruct were then made by inserting the same mutant sequencesdescribed above at the NarI PflmI fragment, and at the PslmI-NcoI sitesdescribed above.

For eukaryotic expressions, these various cDNA constructs bearing wildtype and mutant sequences described above were cloned into theexpression vector pzeoSV (invitrogen). For prokaryotic expression, twoconstructs have been made using the glutathione S-transferase fusionvector pGEX-kg. The inserts which have been attached to the GST fusionnucleotide sequence are the same nucleotide sequence described above(generated with the oligonucleotide primers 969, SEQ ID NO: 139 and 970,SEQ ID NO: 140) bearing either the normal open reading frame nucleotidesequence, or bearing a combination of single and double mutations asdescribed above. This construct allows expression of the full-lengthprotein in mutant and wild type variants in prokaryotic cell systems asa GST fusion protein which allows purification of the full lengthprotein followed by removal of the GST fusion product by thrombindigestion. The second prokaryotic cDNA construct was generated to createa fusion protein with the same vector, and allows the production of theamino acid sequence corresponding to the hydrophilic acidic loop domainbetween TM6 and TM7 of the full-length protein, as either a wild typenucleotide sequence (thus a wild type amino acid sequence for fusionproteins) or as a mutant sequence bearing either the Ala285Val mutation,or the Leu286Val mutation, or the Leu392Val mutation. This wasaccomplished by recovering wild type or mutant sequence from appropriatesources of RNA using the oligonucleotide primers 989:ggatccggtccacttcgtatgctg, SEQ ID NO: 141, and 990:ttttttgaattcttaggctatggttgtgttcca, SEQ ID NO: 142. This allows cloningof the appropriate mutant or wild type nucleotide sequence correspondingto the hydrophilic acid loop domain at the BamHI and the EcoRI siteswithin the pGEX-KG vector.

These prokaryotic expression systems allow the holo-protein or variousimportant functional domains of the protein to be recovered as fusionproteins and then used for binding studies, structural studies,functional studies, and for the generation of appropriate antibodies.

Expression of the ARMP gene in heterologous cell systems can be used todemonstrate structure-function relationships. Ligating the ARMP DNAsequence into a plasmid expression vector to transfect cells is a usefulmethod to test the proteins influence on various cellular biochemicalparameters. Plasmid expression vectors containing either the entire,normal or mutant human or mouse ARMP sequence or portions thereof, canbe used in vitro mutagenesis experiments which will identify portions ofthe protein crucial for regulatory function.

The DNA sequence can be manipulated in studies to understand theexpression of the gene and its product, to achieve production of largequantities of the protein for functional analysis, for antibodyproduction, and for patient therapy. The changes in the sequence may ormay not alter the expression pattern in terms of relative quantities,tissue-specificity and functional properties. Partial or full-length DNAsequences which encode for the ARMP protein, modified or unmodified, maybe ligated to bacterial expression vectors. E. coli can be used using avariety of expression vector systems, e.g., the T7 RNApolymerase/promoter system using two plasmids or by labeling ofplasmid-encoded proteins, or by expression by infection with M13 PhagemGPI-2. E. coli vectors can also be used with Phage lamba regulatorysequences, by fusion protein vectors (e.g. lacZ and trpe), bymaltose-binding protein fusions, and by glutathione-S-transferase fusionproteins, etc., all of which together with many other prokaryoticexpression systems are widely available commercially.

Alternatively, the ARMP protein can be expressed in insect cells usingbaculoviral vectors, or in mammalian cells using vaccinia virus orspecialized eukaryotic expression vectors. For expression in mammaliancells, the cDNA sequence may be ligated to heterlogous promoters, suchas the simian varus (SV40) promoter in the pSV2 vector and other similarvectors and introduced into cultured eukaryotic cells, such as COS cellsto achieve transient or long-term expression. The stable integration ofthe chimeric gene construct may be maintained in mammalian cells bybiochemical selection, such as neomycin and mycophoenolic acid.

The ARMP DNA sequence can be altered using procedures such asrestriction enzyme digestion, fill-in with DNA polymerase, deletion byexonuclease, extension by terminal deoxynucleotide transferase, ligationof synthetic or cloned DNA sequences and site-dDirected sequencealteration with the use of specific oligonucleotides together with PCR.

The cDNA sequence or portions thereof, or a mini gene consisting of acDNA with an introl and its own promoter, is introduced into eukaryoticexpression vectors by conventional techniques. These vectors permit thetranscription of the cDNA in eukaryotic cells by providing regulatorysequences that initiate and enhance the transcription of the cDNA andensure its proper splicing and polyadenylation. The endogenous ARMP genepromoter can also be used. Different promoters within vectors havedifferent activities which alters the level of expression of the cDNA.In addition, certain promoters can also modulate function such as theglucocorticoid-responsive promoter from the mouse mammary tumor virus.

Some of the vectors listed contain selectable markers or neo bacterialgenes that permit isolation of cells by chemical selection. Stablelong-term vectors can be maintained in cells as episomal, freelyreplicating entities using regulatory elements of viruses. Cell linescan also be produced which have integrated the vector into the genomicDNA. In this manner, the gene product is produced on a continuous basis.

Vectors are introduced into recipient cells by various methods includingcalcium phosphate, strontium phosphate, electroporation, lipofection,DEAE dextran, microinjection, or by protoplast fusion. Alternatively,the cDNA can be introduced by infection using viral vectors.

Using the techniques mentioned, the expression vectors containing theARMP gene or portions thereof can be introduced into a variety ofmammalian cells from other species or into non-mammalian cells.

The recombinant cloning vector, according to this invention, comprisesthe selected DNA of the DNA sequences of this invention for expressionin a suitable host. The DNA is operatively linked in the vector to anexpression control sequence in the recombinant DNA molecule so thatnormal and mutant ARMP protein can be expressed. The expression controlsequence may be selected from the group consisting of sequences thatcontrol the expression of genes of prokaryotic or eukaryotic cells andtheir viruses and combinations thereof. The expression control sequencemay be selected from the group consisting of the lac system, the trpsystem, the tac system, the trc system, major operator and promoterregions of phage lambda, the control region of the fd coat protein,early and late promoters of SV40, promoters derived from polyoma,adenovirus, retrovirus, baculovirus, simian virus, 3-phosphoglyceratekinase promoter, yeast acid phosphatase promoters, yeast alpha-matingfactors and combinations thereof.

The host cell which may be transfected with the vector of this inventionmay be selected from the group consisting of E. coli, pseudomonas,bacillus subtillus, bacillus stearothermophilus, or other bacili; otherbacteria, yeast, fungi, insect, mouse or other animal, plant hosts, orhuman tissue cells.

For the mutant ARMP DNA sequence similar systems are employed to expressand produce the mutant protein.

Antibodies to Detect ARMP

Antibodies to epitopes with the ARMP protein can be raised to provideinformation on the characteristics of the proteins. Generations ofantibodies would enable the visualizations of the proteins in cells andtissues using Western blotting. In this technique, proteins are run onpolyacrylamide gel and then transferred onto nitrocellulose membranes.These membranes are then incubated in the presence of the antibody(primary), then following washing antibody which is usedincubated to asecondary detection of the protein-primary antibody complex. Followingrepeated washing, the entire complex is visualized using colourimetricor chemilumine scent methods.

Antibodies to the ARMP protein also allow for the use ofimmunocytochemistry and immunofluorescence techniques in which theproteins can be visualized directly in cells and tissues. This is mosthelpful in order to establish the subcellular location of the proteinand the tissue specificity of the protein.

In order to prepare polyclonal antibodies, fusion proteins containingdefined portions or all of the ARMP protein can be synthesized inbacteria by expression of corresponding DNA sequences in a suitablecloning vehicle. The protein can then be purified, coupled to a carrierprotein and mixed with Freund's adjuvant (to help stimulate theantigenic response by the rabbits) and injected into rabbits or otherlaboratory animals. Alternatively, protein can be isolated from culturedcells expressing the protein. Following booster injections at bi-weeklyintervals, the rabbits or other laboratory animals are then bled and thesera isolated. The sera can be used directly or purified prior to use,by various methods including affinity chromatography, ProteinA-Sepharose, Antigen Sepharose, Anti-mouse-Ig-Sepharose. The sera canthen be used to probe protein extracts run on a polyacrylamide gel toidentify the ARMP protein. Alternatively, synthetic peptides can be madeto the antigenic portions of the protein and used to inoculate theanimals.

To produce monoclonal ARMP antibodies, cells actively expressing theprotein are cultured or isolated from tissues and the cell membranesisolated. The membranes, extracts or recombinant protein extracts,containing the ARMP protein, are injected in Freund's adjuvant intomice. After being injected 9 times over a three week period, the micespleens are removed and resuspended in a phosphate buffered saline(PBS). The spleen cells serve as a source of lymphocytes, some of whichare producing antibody of the appropriate specificity. These are thenfused with a permanently growing myeloma partner cell, and the productsof the fusion are plated into a number of tissue culture wells in thepresence of a selective agent such as HAT. The wells are then screenedto identify those containing cells making useful antibody by ELISA.These are then freshly plated. After a period of growth, these wells areagain screened to identify antibody-producing cells. Several cloningprocedures are carried out until over 90% of the wells contain singleclones which are positive for antibody production. From this procedure astable line of clones is established which produce the antibody. Themonoclonal antibody can then be purified by affinity chromatographyusing Protein A Sepharose, ion-exchange chromatography, as well asvariations and combinations of these techniques.

In situ hybridization is another method used to detect the expression ofARMP protein. In situ hybridization relies upon the hybridization of aspecifically labeled nucleic acid probe to the cellular RNA inindividual cells or tissues. Therefore, it allows the identification ofmRNA within intact tissues, such as the brain. In this method,oligonucleotides corresponding to unique portions of the ARMP gene areused to detect specific mRNA species in the brain.

In this method a rat is anesthetized and transcardially perfused withcold PBS, followed by perfusion with a formaldehyde solution. The brainor other tissues is then removed, frozen in liquid nitrogen, and cutinto thin micron sections. The sections are placed on slides andincubated in proteinase K. Following rinsing in DEP, water and ethanol,the slides are placed in prehybridization buffer. A radioactive probecorresponding to the primer is made by nick translation and incubatedwith the sectioned brain tissue. After incubation and air drying, thelabeled areas are visualized by autoradiography. Dark spots on thetissue sample indicate hybridization of the probe with brain mRNA whichdemonstrates the expression of the protein.

Antibodies may also be used coupled to compounds for diagnostic and/ortherapeutic uses such as radionuclides for imaging and therapy andliposomes for the targeting of compounds to a specific tissue location.

Isolation and Purification of E5-1 Protein

The E5-1 protein may be isolated and purified by 5 the types of methodsdescribed above for the ARMP protein.

The protein may also be prepared by expression of the E5-1 cDNAdescribed herein in a suitable host. The protein is a preferablyexpressed as a fusion protein by ligating its encoding cDNA sequence toa vector containing the coding sequence for another suitable peptide,e.g., GST. The fusion protein is expressed and recovered fromprokaryotic cells such as bacterial or baculovirus cells or fromeukaryotic cells. Antibodies to ARMP, by virtue of portions of aminoacid sequence identity with E5-1, can be used to purify, attract andbind to E5-1 protein and vice versa.

Transgenic Mouse Model of E5-1 Related Alzheimer's Disease

An animal model of Alzheimer's Disease related to mutations of the E5-1gene may be created by methods analogous to those described above forthe ARMP gene.

Antibodies

Due to its structural similarity with the ARMP, the E5-1 protein may beused for the development of probes, peptides, or antibodies to variouspeptides within it which may recognize both the E5-1 and the ARMP geneand gene products, respectively. As a protein homologue for the ARMP,the E5-1 protein may be used as a replacement for a defective ARMP geneproduct. It may also be used to elucidate functions of the ARMP gene intissue culture and vice versa.

Screening for Alzheimer's Disease Linked to Chromosome 1

Screening for Alzheimer's Disease linked to mutations of the E5-1 genemay now be conveniently carried out.

General screening methods are described above in relation to thedescribed mutations in the ARMP gene. These described methods can bereadily applied and adapted to detection of the described chromosome 1mutations, as will be readily understood by those skilled in the art.

In accordance with one embodiment of the invention, the Asn141Ilemutation is screened for by PCR amplification of the surrounding DNAfragment using the primers:

SEQ ID NO: 163 1041: 5′-cattcactgaggacacacc (end-labelled) andSEQ ID NO: 164 1042: 5′-tgtagagcaccaccaaga (unlabelled).

Any tissue with nucleated cell may be examined. The amplified productsare separated by electrophoresis and an autoradiogram of the gel isprepared and examined for mutant bands.

In accordance with a further embodiment, the Met239Val mutation isscreened for by PCR amplification of its surrounding DNA fragment usingthe primers:

1034: 5′-gcatggtgtgcatccact SEQ ID NO: 165 and1035: 5′-ggaccactctgggaggta. SEQ ID NO: 166

The amplified products are separated and an autoradiogram prepared asdescribed above to detect mutant bands.

The same primer sets may be used to detect the mutations by means ofother methods such as SSCP, chemical cleavage, DGGE, nucleotidesequencing, ligation chain reaction and allele specificoligonucleotides. As will be understood by those skilled in the art,other suitable primer pairs may be devised and used.

In inherited cases, as the primary event, and in non-inherited cases asa secondary event due to the disease state, abnormal processing of E5-1,ARMP, APP or proteins reacting with E5-1, APP or ARMP, may occur. Thiscan be detected as abnormal phosphorylation, glycoslyation, glycationamidation or proteolytic cleavage products in body tissues or fluids,e.g, CSF or blood.

Therapies

An important aspect of the biochemical studies using the geneticinformation of this invention is the development of therapies tocircumvent or overcome the ARMP gene defect, and thus prevent, treat,control serious symptoms or cure the disease. In view of expression ofthe ARMP gene in a variety of tissues, one has to recognize thatAlzheimer's Disease may not be restricted to the brain. Alzheimer'sDisease manifests itself as a neurological disorder which in one of itsforms is caused by a mutation in the ARMP gene, but such manifest may becaused by mutations in other organ tissues, such as the liver, releasingfactors which affect the brain activity and ultimately cause Alzheimer'sDisease. Hence, in considering various therapies, it is understood thatsuch therapies may be targeted at tissue other than the brain, such asheart, placenta, lung, liver, skeletal muscle, kidney and pancreas,where ARMP is also expressed.

The effect of these mutations in E5-1 and ARMP is a gain of novelfunction which causes aberrant processing of (APP) Amyloid PrecursorProtein into Aβ peptide, abnormal phosphorylation homeostasis, andabnormal apoptosis. Therapy to reverse this will be small molecules(drugs) recombinant proteins, etc. which block the aberrant function byaltering the structure of the mutant proteins, etc. which block theaberrant function by altering the structure of the mutant protein,enhancing its metabolic clearance or inhibiting binding of ligands tothe mutant protein, enhancing its metabolic clearance or inhibitingbinding of ligands to the mutant protein, or inhibiting the channelfunction of the mutant protein. The same effect might be gained byinserting a second mutant protein by gene therapy similar to thecorrection of the “Deg 1(d)” and “Mec 4(d)” mutations in C. elegans byinsertion of mutant transgenes. Alternatively over expression of wildtype E5-1 protein or wild type ARMP or both may correct the defect. Thiscould be the administration of drugs or proteins to induce thetranscription and translation or inhibit the catabolism of the nativeE5-1 and ARMP proteins. It could also be accomplished by infusion ofrecombinant proteins or by gene therapy with vectors causing expressionof the normal protein at a high level.

Rationale for Therapeutic, Diagnostic, and Investigational Applicationsof the ARMP Gene and Gene Products as They Relate to the AmyloidPrecursor Protein

The Aβ peptide derivatives of APP are neurotoxic (Selkoe et al, 1994).APP is metabolized by passages through the Golgi network and then tosecretory pathways via clathrin-coated vesicles with subsequent passageto the plasma membrane where the mature APP is cleaved by α-secretase toa soluble fraction (Protease Nexin II) plus a non-amyloidogenicC-terminal peptide (Selkoe et al. 1995, Gandy et al., 1993).Alternatively, mature APP can be directed to the endosome-lysosomepathway where it undergoes beta and gamma secretase cleavage to producethe Aβ peptides. The phosphorylation state of the cell determines therelative balance of α-secretase (non-amyloidogenic) or Aβ pathways(amyloidogenic pathway) (Gandy et al., 1993). The phosphorylation stateof the cell can be modified pharmacologically by phorbol esters,muscarinic agonists and other agents, and appears to be mediated bycytosolic factors (especially protein kinase C) acting upon an integralmembrane protein in the Golgi network, which we propose to the ARMP, andmembers of the homologous family (all of which carry severalphosphorylation consensus sequences for protein kinease C). Mutations inthe ARMP gene will cause alterations in the structure and function ofthe ARMP gene product leading to defective interactions with regulatoryelements (e.g., protein kinase C) or with APP, thereby promoting APP tobe directed to the amyloidogenic endosome-lysosome pathway.Environmental factors (viruses, toxins, and aging, etc.) may also havesimilar effects on ARMP. To treat Alzheimer's Disease, thephosphorylation state of ARMP can be altered by chemical and biochemicalagents (e.g. drugs, peptides and other compounds) which alter theactivity of protein kinase C and other protein kinase, or which alterthe activity of protein phosphatases, or which modify the availabilityof ARMP to be postranslationally modified. The interactions betweenkinases and phosphatases with the ARMP gene products (and the productsof its homologues), and the interactions of the ARMP gene products withother proteins involved in the trafficking of the APP within the Golginetwork can be modulated to decrease trafficking of Golgi vesicles tothe endosome-lysosome pathway thereby promoting AP peptide production.Such compounds will include: peptide analogues of APP, ARMP, andhomologues of ARMP as well as other interacting proteins, lipids,sugars, and agents which promote differential glycosylation Of ARMP andits homologues; agents which alter the biologic half-life of messengerRNA or protein of ARMP and homologues including antibodies and antisenseoligonucleotides; and agents which act upon ARMP transcription.

The effect of these agents in cell lines and whole animals can bemonitored by monitoring: transcription; translation; post-translationalmodification of ARMP (e.g., phosphorylation or glycoslyation); andintracellular trafficking of ARMP and its homologues through variousintracellular and extracellular compartments. Methods for these studiesinclude Western and Northern blots; immunoprecipitation after metaboliclabeling (pulse-chase) with radio-labeled methionine and ATP, andimminohistochemistry. The effect of these agents can also be monitoredusing studies which examine the relative binding affinities and relativeamounts of ARMP gene products in interactions with protein kinease Cand/or APP using either standard binding affinity assays orco-precipitation and Western blots using antibodies to protein kineaseC, APP or ARMP and its homologues. The effect of these agents can alsobe monitored by assessing the production of AP peptides by ELISA beforeand after exposure to the putative therapeutic agent (Huang et al.,1993). The effect can also be monitored by assessing the viability ofcell lines after exposure to aluminum salts and to AP peptides which arethrough to be neurotoxic in Alzheimer's Disease. Finally, the effect ofthese agents can be monitored by assessing the cognitive function ofanimals bearing: their normal genotype at APP or ARMP homologues;bearing human APP transgenes (with or without mutations); or bearinghuman ARMP transgenes (with or without mutations); or a combination ofall of these.

Rationale for Therapeutic, Diagnostic, and Investigational Applicationsof the ARMP Gene, the E5-1 Gene and their Products

The ARMP gene product and the E5-1 gene product have amino acid sequencehomology to human ion channel proteins and receptors. For instance, theE5-1 protein shows substantial homology to the human sodium channelα-subunit (E=0.18, P=0.16, identities=22-27% over two regions of atleast 35 amino acid residues) using the BLASTP paradigm of Atschul etal. 1990. Other diseases (such as malignant hyperthermia andhyperkalemic periodic paralysis in humans and the neurodegenerative ofmechanosensory neurons in C. elegans) arise through mutations in ionchannels or receptor proteins. Mutation of the ARMP gene or the E5-1gene could affect similar functions and lead to Alzheimer's Disease andother psychiatric and neurological diseases. Based upon this, a test forAlzheimer's Disease can be produced to detect an abnormal receptor or anabnormal ion channel function related to abnormalities that are acquiredor inherited in the ARMP gene and its product or in one of thehomologous genes such as E5-1 and their products. This test can beaccomplished either in vivo or in vitro by measurements of ion channelfluxes and/or transmembrane voltage or current fluxes using patch clamp,voltage clamp and fluorescent dyes sensitive to intracellular calcium ortransmembrane voltage. Defective ion channel or receptor function canalso be assayed by measurements of activation of second messengers suchas cyclic AMP, cGMP tyrosine kinases, phosphates, increases inintracellular Ca²⁺ levels, etc. Recombinantly made proteins may also bereconstrued in artificial membrane systems to study ion channelconductance. Therapies which affect Alzheimer's Disease (due toacquired/inherited defects in the ARMP gene or E5-1 gene; due to defectsin other pathways leading to this disease such as mutations in APP; anddue to environmental agents) can be tested by analysis of their abilityto modify an abnormal ion channel or receptor function induced bymutation in the ARMP gene or in one of its homologues. Therapies couldalso be tested by their ability to modify the normal function of an ionchannel or receptor capacity of the ARMP gene products and itshomologues. Such assays can be performed on cultured cells expressingendogenous normal or mutant ARMP genes/gene products or E5-1 genes/geneproducts. Such studies can be performed in addition on cells transfectedwith vectors capable of expressing ARMP, parts of the ARMP gene and geneproduct, mutant ARMP, E5-1 gene, parts of the E5-1 gene and geneproduct, mutant E5-1 gene or another homologue in normal or mutant form.Therapies for Alzheimer's Disease can be devised to modify an abnormalion channel or receptor function of the ARMP gene or E5-1 gene. Suchtherapies can be conventional drugs, peptides, sugars, or lipids, aswell as antibodies or other ligands which affect the properties of theARMP or E5-1 gene product. Such therapies can also be performed bydirect replacement of the ARMP gene and/or E5-1 gene by gene therapy. Inthe case of an ion channel, the gene therapy could be performed usingeither mini-genes (cDNA plus a promoter) or genomic constructs bearinggenomic DNA sequences for parts or all of the ARMP gene. Mutant ARMP orhomologous gene sequence might also be used to counter the effect of theinherited or acquired abnormalities of the ARMP gene as has recentlybeen done for replacement of the mec 4 and deg 1 in C. elegans (Huangand Chalfie, 1994) The therapy might also be directed at augmenting thereceptor or ion channel function of the homologous genes such as theE5-1 gene, in order that it may potentially take over the functions ofthe ARMP gene rendered defective by acquired or inherited defects.Therapy using antisense oligonucleotides to block the expression of themutant ARMP gene or the mutant E5-1 gene, coordinated with genereplacement with normal ARMP or E5-1 gene can also be applied usingstandard techniques of either gene therapy or protein replacementtherapy.

Protein Therapy

Treatment of Alzheimer's Disease can be performed by replacing themutant protein with normal protein, or by modulating the function of themutant protein. Once the biological pathway of the ARMP protein has beencompletely understood, it may also be possible to modify thepathophysiologic pathway (e.g., a signal transduction pathway) in whichthe protein participates in order to correct the physiological defect.

To replace the mutant protein with normal protein, or with a proteinbearing a deliberate counterbalancing mutation it is necessary to obtainlarge amounts of pure ARMP protein or E5-1 protein from cultured cellsystems which can express the protein. Delivery of the protein to theaffected brain areas or other tissues can then be accomplished usingappropriate packaging or administrating systems.

Gene Therapy

Gene therapy is another potential therapeutic approach in which normalcopies of the ARMP gene are introduced into patients to successfullycode for normal protein in several different affected cell types. Thegene must be delivered to those cells in a form in which it can be takenup and code for sufficient protein to provide effective function.Alternatively, in some neurologic mutants it has been possible toprevent disease by introducing another copy of the homologous genebearing a second mutation in that gene or to alter mutation, or useanother gene to block its effect.

Retroviral vectors can be used for somatic cell gene therapy especiallybecause of their high efficiency of infection and stable integration andexpression. The targeted cells however must be able to divide and theexpression of the levels of normal protein should be high because thedisease is a dominant one. The full length ARMP gene can be cloned intoa retroviral vector and driven from its endogenous promoter or from theretroviral long terminal repeat or from a promoter specific for thetarget cell type of interest (such as neurons).

Other viral vectors which can be used include adeno-associated virus,vaccinia virus, bovine papilloma virus, or a herpesvirus such asEpstein-Barr virus.

Gene transfer could also be achieved using non-viral means requiringinfection in vitro. This would include calcium phosphate, DEAE dextran,electroporation, and protoplast fusion. Liposomes may also bepotentially beneficial for delivery of DNA into a cell. Although thesemethods are available, many of these are lower efficiency.

Antisense based strategies can be employed to explore ARMP gene functionand as a basis for therapeutic drug design. The principle is based onthe hypothesis that sequence-specific suppression of gene expression canbe achieved by intracellular hybridization between mRNA and acomplementary antisense species. The formation of a hybrid RNA duplexmay then interfere with the processing/transport/translation and/orstability of the target ARMP mRNA. Hybridization is required for theantisense effect to occur, however the efficiency of intracellularhybridization is low and therefore the consequences of such an event maynot be very successful. Antisense strategies may use a variety ofapproaches including the use of antisense oligonucleotides, injection ofantisense RNA and transfection of antisense RNA expression vectors.Antisense effects can be induced by control (sense) sequences, however,the extent of phenotypic changes are highly variable. Phenotypic effectsinduced by antisense effects are based on changes in criteria such asprotein levels, protein activity measurement, and target mRNA levels.Multidrug resistance is a useful model to study molecular eventsassociated with phenotypic changes due to antisense effects, since themultidrug resistance phenotype can be established by expression of asingle gene mdrl (MDR gene) encoding for P-glycoprotein.

Transplantation of normal genes into the affected area of the patientcan also be useful therapy for Alzheimer's Disease. In this procedure, anormal hARMP protein is transferred into a cultivable cell type such asglial cells, either exogenously or endogenously to the patient. Thesecells are then injected serotologically into the disease affectedtissue(s). This is a known treatment for Parkinson's disease.

Immunotherapy is also possible for Alzheimer's Disease. Antibodies canbe raised to a mutant ARMP protein (or portion thereof) and thenadministered to bind or block the mutant protein and its deleteriouseffects. Simultaneously, expression of the normal protein product couldbe encouraged. Administration could be in the form of a one timeimmunogenic preparation or vaccine immunization. An immunogeniccomposition may be prepared as injectables, as liquid solutions oremulsions. The ARMP protein may be mixed with pharmaceuticallyacceptable excipients compatible with the protein. Such excipients mayinclude water, saline, dextrose, glycerol, ethanol and combinationsthereof. The immunogenic composition and vaccine may further containauxiliary substances such as emulsifying agents or adjuvants to enhanceeffectiveness. Immunogenic compositions and vaccines may be administeredparenterally by injection subcutaneously or intramuscularly.

The immunogenic preparations and vaccines are administered in suchamount as will be therapeutically effective, protective and immunogenic.Dosage depends on the route of administration and will vary according tothe size of the host.

Similar gene therapy techniques may be employed with respect to the E5-1gene.

The above disclosure generally describes the present invention. A morecomplete understanding can be obtained by reference to the followingspecific examples. These examples are described solely for purposes ofillustration and are not intended to limit the scope of the invention.Changes in the form and substitution of equivalents are contemplated ascircumstances may suggest or render expedient. Although specific termshave been employed herein, such terms are intended in a descriptivesense and not for purposes of limitations.

Example 1 Development of the Genetic, Physical “contig” andTranscriptional Map of the Minimal Co-Segregating Region

The CEPH Mega YAC and the RPCI PAC human total genomic DNA librarieswere searched for clones containing genomic DNA fragments from the AD3region of chromosome 14q24.3 using oligonucleotide probes for each ofthe ## SSR marker loci used in the genetic linkage studies as well as ##additional markers depicted in FIG. 1 a (Albertsen et al., 1990;Chumakov et al., 1992; Joannu et al., 1994). The genetic map distancesbetween each marker are depicted above the contig, and are derived frompublished data (NIH/CEPH Collaborative Mapping Group, 1992; Wang, 1992;Weissenbach, J. et al., 1992 Gyapay, G et al., 1994). Clones recoveredfor each of the initial marker loci were arranged into an ordered seriesof partially overlapping clones (“contig”) using four independentmethods. First, sequences representing the ends of the YAC insert wereisolated by inverse PCR (Riley et al., 1990), and hybridized to Southernblot panels containing restriction digests of DNA from all of the YACclones bearing overlapping sequences. Second, inter-Alu PCR wasperformed on each YAC, and the resultant band patterns were comparedacross the pool of recovered YAC clones in order to identify otherclones bearing overlapping sequences (Bellamne-Chartelot et al., 1992;Chumakov et al; 1992). Third, to improve the specificity of the Alu-PCRfingerprinting, we restricted the YAC DNA with HaeIII or RsaI, amplifiedthe restriction products with both Alu and L1H consensus primers, andresolved the products by polyacrylamide gel electrophoresis. Finally, asadditional STSs were generated during the search for transcribedsequences, these STSs were also used to identify overlaps. The resultantcontig was complete except for a single discontinuity between YAC932C7bearing D14S53 and YAC746B4 containing D14S61. The physical map order ofthe STSs within the contig was largely in accordance with the geneticlinkage map for this region (NIH/CEPH Collaborative Mapping Group, 1992;Wang, Z., Webber, J. L., 1992; Weissenbach, J. et al., 1992; Gyapay, G.et al., 1994). However, as with the genetic maps, we were unable tounambiguously resolve the relative order of the loci within the D14S43/D 14S71 cluster and the D14S76/D14S273 cluster. PAC I clonessuggest that D14S277 is telomeric to D14S268, whereas genetic maps havesuggested the reverse order. Furthermore, a few STS probes failed todetect hybridization patterns in at least one YAC clone which, on thebasis of the most parsimonious consensus physical map and from thegenetic map, would have been predicted to contain that STS. Forinstance, the D14S268 (AFM265) and RSCAT7 STSs are absent fromYAC788H12. Because these results are reproducible, and occurred withseveral different STS markers, these results most likely reflect thepresence of small interstitial deletions with one of the YAC clones.

Example 2 Cumulative Two-point lod Scores for Chromosome 14q24.3 Markers

Genotypes of each polymorphic microsatellite marker locus weredetermined by PCR from 100 ng of genomic DNA of all available affectedand unaffected pedigree members as previously described (St.George-Hyslop, P et al, 1992) using primer sequences specific for eachmicrosatellite locus (Weissenbach, J et al., 1992; Gyapay, G et al.,1994). The normal population frequency of each allele was determinedusing spouses and other neurologically normal subjects from the sameethnic groups, but did not differ significantly from those establishedfor mixed Caucasian populations (Weissenbach, J. et al., 1992; Gyapay,G. et al., 1994). The maximum likelihood calculations assumed an age ofonset correction, marker allele frequencies derived from publishedseries of mixed Caucasian subjects, an estimated allele frequence forthe AD3 mutation of 1:1000 as previously described (St. George-Hyslop,P. et al., 1992). The analyses were repeated using equal marker allelefrequencies, and using phenotype information only from affected pedigreemembers as previously described to ensure that inaccuracies in theestimated parameters used in the maximum likelihood calculations did notmisdirect the analyses (St. George-Hyslop, P. et al., 1992). Thesesupplemental analyses did not significantly alter either the evidencesupporting linkage, or the discovery of recombination events.

Example 3 Haplotypes Between Flanking Markers Segregated with AD3 in FADPedigrees

Extended haplotypes between the centromeric and telomeric flankingmarkers on the parental copy of chromosome 14 segregating with AD3 infourteen early onset FAD pedigrees (pedigrees NIH2, MGH 1, Tor1.1, FAD4,FAD 1, MEX1, and FAD2 show pedigree specific lod scores ≧+3.00 with atleast one marker between D14S258 and D14S53). Identical partialhaplotypes (boxed) are observed in two regions of the disease bearingchromosome segregating in several pedigrees of similar ethnic origin. Inregion A, shared alleles are seen at D14S268 (“B”: allele size=126 bp,allele frequence in normal Caucasians=0.04; “C”: size=124 bp,frequency=0.38); D14S277 (“B”: size=156 bp, frequency=0.19; “C” size=154bp, frequency=0.33); and RSCAT6 (“D”: size=111 bp, frequency 0.25; “E”size=109 bp, frequency=0.20; “F” size=107 bp, frequency=0.47). In regionB, alleles of identical size are observed at D14S43 (“A”: size=193 bp,frequency=0.01; “D”: size 187 bp, frequency=0.12; “E” size=185 bp,frequency=0.26; “1” size=160 bp, frequency=0.38); D14S273 (“3”: size=193bp, frequency=0.38; “4” size=191 bp, frequency=0.16; “5”: size=189 bp,frequency=0.34; “6”: size=187 bp, frequency=0.02) and D14S76 (“1”:size=bp, frequency=0.01; “5”: size=bp, frequency=0.38; “6”: size=bp,frequency=0.07, “9”: size=bp, frequency=0.38). The ethnic origins ofeach pedigree are abbreviated as: Ashk=Askenazi Jewish; Ital=SouthernItalian; Angl=Anglo-Saxon-Celt; FrCan=French Canadian; Jpn=Japanese;Mex=Mexican Caucasian; Ger=German; Am=American Caucasian. The type ofmutation detected is depicted by the amino acid substitution andputative condon number or by ND where no mutation has been detectedbecause a comprehensive survey has not been undertaken due to theabsence of a source of mRNA for RT-PCR studies.

Example 4 Recovery of Transcribed Sequences from the AD3 Interval

Putative transcribed sequences encoded in the AD3 interval wererecovered using either a direct hybridization method in which short cDNAfragments generated from human brain mRNA were hybridized to immobilizedcloned genomic DNA fragments (Rommens, J M et al., 1993). The resultantshort putatively transcribed sequences were used as probes to recoverlonger transcripts from human brain cDNA libraries (Stratagene, LaJolla). The physical locations of the original short clone and of thesubsequently acquired longer cDNA clones were established by analysis ofthe hybridization pattern generated by hybridizing the probe to Southernblots containing a panel of EcoRI digested total DNA samples isolatedfrom individual YAC clones within the contig. The nucleotide sequence ofeach of the longer cDNA clones was determined by automated cyclesequencing (Applied Biosystems Inc., CA), and compared to othersequences in nucleotide and protein databases using the blast algorithm(Atschul, S F et al., 1990). Accession numbers for the transcribedsequences in this report are L40391, L40392, L40393, L40394, L40395,L40396, L40397, L40398, L40399, L40400, L40401, L40402, and L40403.

Example 5 Locating Mutations in the ARMP Gene Using Restriction Enzymes

The presence of Ala 246 Glu mutation which creates a Ddel restrictionsite was assayed in genomic DNA by PCR using the end labelled primer 849(5′-atctccggcaggcatatct-3′) SEQ ID NO: 129 and the unlabelled primer 892(5′-tgaaatcacagccaagatgag-3′) SEQ ID NO: 130 to amplify an 84 bp genomicexon fragment using 100 ng of genomic DNA template, 2 mM MgCl₂, 10pMoles of each primer, 0.5U Taq polymerase, 250 uM dNTPs for 30 cyclesof 95° C.×20 seconds, 60° C.×20 seconds, 72° C.×5 seconds. The productswere incubated with an excess of DdeI for 2 hours according to themanufacturers protocol, and the resulting restriction fragments wereresolved on a 6% nondenaturing polyacrylamide gel and visualized byautoradiography. The presence of the mutation was inferred from theclevage of the 84 bp fragment due to the presence of a DdeI restrictionsite. All affected members of the FAD I pedigree (filled symbols) andseveral at-risk members (“R”) carried the DdeI site. None of theobligate escapees (those individuals who do not get the disease, age >70years), and none of the normal controls carried the DdeI mutation.

Example 6 Location Mutation in the ARMP Gene Using Allelle SpecificOligonucleotides

The presence of the Cys 410 Tyr mutation was assayed using allelespecific oligonucleotides. 100 ng of genomic DNA was amplified with theexonic sequence primer 885 (5′-tggaaactggaacacaac-3′) SEQ ID NO: 127 andthe opposing intronic sequence primer 893 (5′-gtgtggccagggtagagaact-3′)SEQ ID NO: 128 using the above reaction conditions except 2.5 mM MgCl₂,and cycle conditions of 94° C.×20 seconds, 58° C.×20 seconds, and 72° C.for 10 seconds). The resultant 216 bp genomic fragment was denatured by10-fold dilution in 0.4M NaOH, 25 mM EDTA, and was vacuum slot-blottedto duplicate nylon membranes. The end-labelled “wild-type” primer 890(5′-ccatagcctgtttcgtagc-3′) SEQ ID NO: 131 and the end-labelled “mutant”primer 891 (5′-ccatagcctatttcgtagc-3′) SEQ ID NO: 132 were hybridized toseparate copies of the slot-blot filters in 5×SSC, 5×Denhardt's, 0.5%SDS for 1 hour at 48° C., and then washed successively in 2×SSC at 23°C. and 2×SSC, 0.1% SDS at 50° C. and then exposed to X-ray film. Alltestable affected members as well as some at-risk members of the AD3(shown) and NIH2 pedigrees (not shown) possessed the Cys 410 Tyrmutation. Attempts to detect the Cys 410 Try mutation by SSCP revealedthat a common intronic sequence polymorphism migrated with the same SSCPpattern.

Example 7 Northern Hybridization Demonstrating the Expression of ARMP 25Protein mRNA in a Variety of Tissues

Total cytoplasmic RNA was isolated from various tissue samples(including heart, brain, and different regions of placenta, lung, liver,skeletal muscle, kidney and pancreas) obtained from surgical pathologyusing standard procedures such as CsCl purification. The RNA was thenelectrophoresed on a formaldehyde gel to permit size fractionation.

The nitrocellulose membrane was prepared and the RNA was thentransferred onto the membrane. ³²P-labelled cDNA probes were preparedand added to the membrane in order for hybridization between the probethe RNA to occur. After washing, the membrane was wrapped in plasticfilm and placed into imaging cassettes containing X-ray film. Theautoradiographs were then allowed to develop for one to several days.The positions of the 28S and 18S rRNA bands are indicated. Sizing wasestablished by comparison to standard RNA markers. Analysis of theautoradiographs revealed a prominent band at 3.0 kb in size. Thesenorthern blots demonstrated the ARMP gene is expressed in all of thetissues examined.

Example 8 Eukaryotic and Prokaryotic Expression Vector Systems

Eukaryotic and prokaryotic expression systems have been generated usingtwo different classes of ARMP nucleotide cDNA sequence inserts. In thefirst class, termed full-length constructs, the entire ARMP cDNAsequence was inserted into the expression plasmid in the correctorientation, and included both the natural 5′ UTR and 3′ UTR sequencesas well as the entire open reading frame. The open reading frames bear anucleotide sequence cassette which allows either the wild type openreading frame to be included in the expression system or alternatively,single or a combination of double mutations can be inserted into theopen reading frame. This was accomplished by removing a restrictionfragment from the wild type open reading frame using the enzymes NarIand PflmI and replacing it with a similar fragment generated by reversetranscriptase PCR and which bears the nucleotide sequence encodingeither the Met 146Leu mutation or the Hys163Arg mutation. A secondrestriction fragment was removed from the wild type normal nucleotidesequence for the open reading frame by cleavage with the enzymes PflmIand NcoI and replaced with restriction fragments bearing either thenucleotide sequence encoding the Ala246Glu mutation, or the Ala260Valmutation or the Ala285Val mutation or the Leu286Val mutation, or theLeu392Val mutation, or the Cys410 Tyr mutation. Finally, a third variantbearing combinations of either the Met 146Leu or His163Arg mutations intandem with the remaining mutations by linking the NarI-PflmI fragmentbearing these mutations and the PflmI-NcoI fragment bearing theremaining mutations.

A second variant of cDNA inserts bearing wild type or mutant cDNAsequences was constructed by removing from the full-length cDNA the 5′UTR and part of the 3′ UTR sequences. The 5′ UTR sequence was replacedwith a synthetic oligonucleotide containing a KpnI restriction site anda Kozak initiation site (oligonucleotide 969:ggtaccgccaccatgacagaggtacctgcac) SEQ ID NO: 139. The 3′ UTR was replacedwith an oligonucleotide corresponding to position 2566 of the cDNA andbears an artificial EcoRI site (oligonucleotide970:gaattcactggctgtagaaaaagac) SEQ ID NO: 140. Mutant variants of thisconstruct were then made by inserting the same mutant sequencesdescribed above at the NarI-PflmI fragment, and at the PslmI-NcoI sitesdescribed above.

For eukaryotic expressions, these various cDNA constructs bearing wildtype and mutant sequences were cloned into the expression vector pZeoSV(invitrogen). For prokaryotic expression, two constructs were made usingthe gluthathione S-transf erase fusion vector pGEX-kg. The inserts whichhave been attached to the GST fusion nucleotide sequence are the samenucleotide sequence described above generated with the oligonucleotideprimers 969, SEQ ID NO: 139 and 970, SEQ ID NO: 140, bearing either thenormal open reading frame nucleotide sequence or bearing a combinationof single and double mutations as described above. This construct allowsexpression of the full-length protein in mutant and wild type variantsin prokaryotic cell systems as a GST fusion protein which will allowpurification of the full-length protein followed by removal of the GSTfusion product by thrombin digestion. The second prokaryotic cDNAconstruct was generated to create a fusion protein with the same vector,and allows the production of the amino acid sequence corresponding tothe hydrophillic acid loop domains between TM6 and TM7 of thefull-length protein, as either a wild type nucleotide sequence (thus awild type amino acid sequence for fusion proteins) or as a mutantsequence bearing either the Ala285Val mutation, or the Leu286Valmutation, or the Leu392Val mutation. This was accomplished by recoveringwild type or mutant sequence from appropriate sources of RNA using theoligonucleotide primers 989:ggatccggtccacttcgtatgctg SEQ ID NO: 141, and990:ttttttgaattcttaggctatggttgtgttcca SEQ ID NO: 142. This allowscloning of the appropriate mutant or wild type nucleotide sequencecorresponding to the hydrophillic acid loop domain at the BamHI and theEcoRI sites within the pGEX-KG vector.

These prokaryotic expression systems allow the holo-protein or variousimportant functional domains of the protein to be recovered as fusionproteins and then used for binding studies, structural studies,functional studies, and for the generation of appropriate antibodies.

Example 9 Identification of Three New Mutations in the ARMP Gene

Three novel mutations have been identified in subjects affected withearly onset Alzheimer's Disease. All of these mutations co-segregatewith the disease, and are absent from at least 200 normal chromosomes.The three mutations are as follows: a substitution of C by T at position1027 which results in the substitution of alanine 260 for valine;substitution of C by T at position 1102, which results in thesubstitution of alanine at 285 by valine; and substitution of C by G atposition 1422 which results in the substitution of leucine 392 byvaline. Significantly, all of these mutations occur within the acidichydrophillic loop between putative TM6 and TM7. Two of the mutations(A260V; A285V) and the L286V mutation are also located in thealternative spliced domain.

The three new mutations, like the other mutations, can be assayed by avariety of strategies (direct nucleotide sequencing, Allele specificoligos, ligation polymerase chain reaction, SSCP, RFLPs) using RT-PCRproducts representing the mature mRNA/cDNA sequence or genomic DNA. Wehave chosen allele specific oligos. For the A260V and the A285Vmutations, genomic DNA carrying the exon can be amplified using the samePCR primers and methods for the L286V mutation. PCR products were thendenatured and slot blotted to duplicate nylon membranes using the slotblot protocol described for the C410T mutation.

The Ala260Val mutation was scored by these blots by using hybridizationwith end-labeled allele-specific oligonucleotides corresponding to thewild type sequence (994:gattagtggttgttttgtg) SEQ ID NO: 143 or themutant sequence (995:gattagtggctgttttgtg) SEQ ID NO: 144 byhybridization at 48° C. followed by a wash at 52° in 3×SSC buffercontaining 0.1% SDS. The Ala285Val mutation was scored on these slotblots as described above but using instead the allele-specificoligonucleotides for the wild type sequence (1003:tttttccagctctcattta)SEQ ID NO: 145 or the mutant primer (1004:tttttccagttctcattta) SEQ IDNO: 146 at 48° C. followed by washing at 52° C. as above except that thewash solution was 2×SSC.

The Leu392Val mutation was scored by amplification of the exon fromgenomic DNA using primers 996 (aaacttggattgggagat) SEQ ID NO: 167 and893 (gtgtggccagggtagagaact) SEQ ID NO: 128 using standard PCR bufferconditions excepting that the magnesium concentration was 2 mM and cycleconditions were 94° C. times 10 seconds, 56° C. times 20 seconds, and72° C. for 10 seconds. The result 200 based pair genomic fragment wasdenatured as described for the Cys410Tyr mutation and slot-blotted induplicate to nylon membranes. The presence or absence of the mutationwas then scored by differential hybridization to either a wild typeend-labelled oligonucleotide (999:tacagtgttctggttggta) SEQ ID NO: 148 orwith an end-labeled mutant primer (100:tacagtgttgtggttggta) SEQ ID NO:149 by hybridization at 45° C. and then successive washing in 2×SSC at23° and then at 68° C.

Example 10 Polyclonal Antibody Production

Peptide antigens were synthesized by solid-phase techniques and purifiedby reverse phase high pressure liquid chromatography. Peptides werecovalently linked to keyhole limpet hematoxylin (KLH) via disulfidelinkages that were made possible by the addition of a cystein residue atthe peptide C-terminus. This additional residue does not appear normallyin the protein sequence and was included only to facilitate linkage tothe KLH molecule. A total of three rabbits were immunized withpeptide-KLH complexes for each peptide antigen and were thensubsequently giveN booster injections at seven day intervals. Antiserawere collected for each peptide and pooled and IgG precipitated withammonium sulfate. Antibodies were then affinity purified with Sulfa-linkagarose (Pierce) coupled with the appropriate peptide. This finalpurification is required to remove non-specific interactions of otherantibodies present in either the pre- or post-immune serum.

The specific sequences to which we have raised antibodies are:

Polyclonal antibody 1: SEQ ID NO: 169 NDNRERQEHNDRRSL (C)-residues 30-44Polyclonal antibody 2: SEQ ID NO: 170KDGQLIYTPFTEDTE (C)-residues 109-123 Polyclonal antibody 3:SEQ ID NO: 171 EAQRRVSKSKYNAE (C)-residues 304-318

The non-native cysteine residue is indicated at the C-terminal by (C).These sequences are contained within various predicted domains of theprotein. For example, antibodies 1, 3, and 4 are located in potentiallyfunctional domains that are exposed to the aqueous media and may beinvolved in binding to other proteins critical for the development ofthe disease phenotype. Antibody 2 corresponds to a short linking regionsituated between the predicted first and second transmembrane helices.

Example 11 Identification of Two Mutations in E5-1 Gene

RT-PCR products corresponding to the E5-10RF were generated from RNA oflymphoblasts or frozen post-mortem brain tissue using oligonucleotideprimer pairs 1021:5′-cagaggatggagagaatac SEQ ID NO: 172 and1018:5′-ggctccccaaaactgtcat SEQ ID NO:173 (product=888 bp); and1071:5′-gccctagtgttcatcaagta SEQ ID NO: 174 and1022:5′-aaagcgggagccaaagtc SEQ ID NO: 175 (product=826 bp) by PCR using250 iMol dNTPs, 2.5 mM MgCl2, IOpMol oligunucleotides in 10 μl cycledfor 40 cycles of 94° C.×20 seconds, 58° C.×20 seconds, 72° C.×45seconds. The PCR products were sequenced by automated cycle sequencing(ABI, Foster City, A) and the fluorescent chromatograms were scanned forheterozygous nucleotide substitutions by direct inspection and by theFactura (ver 1.2.0) and Sequence Navigator (ver 1.0.1b15) softwarepackages (data not shown).

Asn141Ile: the A→T substitution at nucleotide 787 creates a BclIrestriction site. The exon bearing this mutation was amplified from 100ng of genomic DNA using 10pMol of oligonucleotides 1041:5′-cattcactaggacacacc SEQ ID NO: 163 (end-labelled) and 1042:5′-tgtagagcaccaccaaga SEQ ID NO: 164 (unlabelled), and PCR reactionconditions similar to those described below for the Met239Val. 2 μl ofthe PCR product was restricted to Bc1I (NEBL, Beverly, Mass.) in 10 μlreaction volume according to the manufacturers' protocol, and theproducts were resolved by non-Denaturing polyacrylamide gelelectrophoresis. In subjects with wild type sequences, the 114 bp PCRproduct is cleaved into 68 bp and 46 bp fragments. Mutant sequencescause the product to be cleaved into 53 bp, 46 bp and 15 bp.

Met239Val: The A-4G substitution at nucleotide 1080 deletes a NlaIIIrestriction site, allowing the presence of the Met239Val mutation to bedetected by amplification from 100 ng of genomic DNA using PCR (10 pMololigonucleotides 1034:5′-gcatggtgtgcatccact SEQ ID NO: 165,1035:5′-ggaccactctgggaggta SEQ ID NO: 166; 0.5 U Taq polymerase, 250 αμMdNTPS, 1 μCi alpha ³²P-DCTP, 1.5 mM MgCl₂, 101 volume; 30 cycles of 94°C. X30 seconds, 58° C.×20 seconds, 72° C.×20 seconds) to generate a 110bp product. 2 μl of the PCR reaction were diluted to 10 gl andrestricted with 3 U of NlaIII (NEBL, Beverly Mass.) for 3 hours. Therestriction products were resolved by non-denaturing polyacrylamide gelelectrophoresis and visualized by autoradiography. Normal subjects showcleavage products of 55, 35, 15 and 6 bp, whereas the mutant sequencegives fragments of 55, 50 and 6 bp.

Although preferred embodiments of the invention have been describedherein in detail, it will be understood by those skilled in the art thatvariations may be made thereto without departing from the spirit of theinvention or the scope of the appended claims.

TABLE 1 RECOMBINATION FRACTION (O) LOCUS 0.00 0.05 0.10 0.15 0.20 0.300.40 D 14S63 -∞ 1.54 3.90 4.38 4.13 2.71 1.08 D 14S258 -∞ 21.60 19.6417.19 14.50 8.97 3.81 D 14S77 -∞ 15.18 15.53 14.35 12.50 7.82 2.92 D14S71 -∞ 15.63 14.14 12.19 10.10 5.98 2.39 D 14S43 -∞ 19.36 17.51 15.2712.84 7.80 3.11 D 14S273 -∞ 12.30 11.52 10.12 8.48 5.04 1.91 D 14S61 -∞26.90 24.92 22.14 18.98 12.05 5.07 D 14S53 -∞ 11.52 11.41 10.39 8.995.73 2.51 D 14S48 -∞ 0.50 1.05 1.14 1.04 0.60 0.18

TABLE 2 PEDIGREE ID

TABLE 3 No. 1 Target File marmp.con/long [Frame 1] Key 1 Target 1Overlap 467 Match 465 Similarities Percentage 99.57%   1       10        20        30        40        50        60        70HumanN- MTELPAPLSYFQNAQMSEDNHLSNTVRSQNDNRERQEHNDRRSLGHPEPLSNGRPQGNSRQVVEQDEEEDSEQ ID NO: 2   **********************************************************************MouseN- MTEIPAPLSYFQNAQMSEDSHSSSAIRSQNDSQERQQQHDRQRLDNPEPISNGRPQSNSRQVVEQDEEEDSEQ ID NO: 4   1       10        20        30        40        50        60        70  71       80        90       100       110       120       130       140   EELTLKYGAKHVIMLFVPVTLCMVVVVATIKSVSFYTRKDGQLIYTPFTEDTETVGQRALHSILNAAIMI   **********************************************************************   EELTLKYGAKHVIMLFVPVTLCMVVVVATIKSVSFYTRKDGQLIYTPFTEDTETVGQRALHSILNAAIMI  71       80        90       100       110       120       130       140 141      150       160       170       180       190       200       210   SVIVVMTILLVVLYKYRCYKVIHAWLIISSLLLLFFFSFIYLGEVFKTYNVAVDYITVALLIWNLGVVGM   **********************************************************************   SVIVTMTILLVVLYKYRCYKVIHAWLIISSLLLLFFFSFIYLGEVFKTYNVXVDYVTVALLIWNWGVVGM 141      150       160       170       180       190       200       210 211      220       230       240       250       260       270       280   ISIHWKGPLRLQQAYLIMISALMALVFIKYLPEWTAWLILAVISVYDLVAVLCPKGPLRMLVETAQERNE   **********************************************************************   IAIHWKGPLRLQQAYLIMISALMALVFIKYLPEWTAWLILAVISVYDLVAVLCPKGPLRMLVETAQERNE 211      220       230       240       250       260       270       280 281      290       300       310       320       330       340       350   TLFPALIYSSTMVWLVNMAEGDPEAQRRVSKNSKYNAESTERESQDTVAENDDGGFSEEWEAQRDSHLGP   **********************************************************************   TLFPALIYSSTMVWLVNMAEGDPEAQRRVPKNPKYNTQRAERETQDSGSGNDDGGFSEEWEAQRDSHLGP 281      290       300       310       320       330       340       350 351      360       370       380       390       400       410       420   HRSTPESRAAVQELSSSILAGEDPEERGVKLGLGDFIFYSVLVGKASATASGDWNTTIACFVAILIGLCL   **********************************************************************   HRSTPESRAAVQELSGSILTSEDPEERGVKLGLGDFIFYSVLVGKASATASGDWNTTIACXVAILIGLCL 351      360       370       380       390       400       410       420 421      430       440       450       460   TLLLLAIFKKALPALPISITFGLVFYFATDYLVQPFMDQLAFHQFYI -C   ***********************************************   XLLLLAIYKKGXPAXPISITFGFVFXFATDYLVQPFMDQLAFHQFYI -C 421      430       440       450       460

TABLE 4 HUMAN ARMP FUNCTION DOMAINS Domains (Amino Acid Residue)Functional Characteristic  82-100 AA Hydrophobic 132-154 AA Hydrophobic164-183 AA Hydrophobic 195-213 AA Hydrophobic 221-238 AA Hydrophobic244-256 AA Hydrophobic 281-299 AA Hydrophobic 404-428 AA Hydrophobic431-449 AA Hydrophobic 115-119 AA (YTPF) SEQ ID NO: 161 PhosphorylationSite 353-356 AA (STPC) SEQ ID NO: 162 Phosphorylation Site 300-385 AAAcid Rich Domain Possible Metal Binding Domain ANTIGENIC SITES INCLUDINGAMINO ACID RESIDUES 27-44 46-48 50-60 66-67 107-111 120-121 125-126155-160 185-189 214-223 220-230 240-245 267-269 273-282 300-370 400-420

TABLE 5 ENZYME AMPLIFICATION AMPLIFICATION (effect of 0440 0440ALLELE-SPECIFIC MUTATION mutation) #1 #2 0440 M 146LEU Bsphl910 (170-S182F) 911 (170-S182)R (destroy) TCACAGAAGATACCGCCCAACCATAAGAAG AGACT AACAG (SEQ ID NO: 176) (SEQ ID NO: 177)MIS 164 Ary Nla III 927 (intronic) 928 (destroy) TCTGTACTTTTTAAGACTTCAGAGTAATTC GGTTGTG ATCANCA (SEQ ID NO: 178) (SEQ ID NO: 179)Ala 246 Dlc I 849* 892 (create) GACTCCAGCAGGCAT TGAAATCACAGCCAA ATCTGATGAG (SEQ ID NO: 80) (SEQ ID NO: 130) Leu 286 Val Pvu II 952 951(create) GATGAGACAAGTNCC CACCCATTTACAAGT NTGAA TTAGC (SEQ ID NO: 181)(SEQ ID NO: 183) 945 TTAGTGGCTGTTTNG TGTCC (SEQ ID NO: 82) Cys 410 TysAllele speci- 823 885 CCATAGCCTGTTTCGTAGC fic ligo GTGTGGCCAGGGTAGTGGAGACTGGAACAC (SEQ ID NO: 131) AGAACT AAC 890 = WT (SEQ ID NO: 128)(SEQ ID NO: 127) CCATAGCCTATTTCGTAGC (SEQ ID NO: 132) 891 = MUT

TABLE 6 POSITION OF EXONS AND INTRON-EXON BOUNDARIES OF THE ARMP GENEcDNA/mRNA SEQUENCE CORRESPONDING GENOMIC SEQUENCE Transcript ID Genomicsequence file ARMP (917 ver) CC44 ver ID & position of exon Comments1-113 bp N/A 917-936.gen @ 731-834 bp Alternate 5′UTR N/A 1-422 bp917-936.gen @ 1067-1475 bp Alternate 5′UTR 114-195 bp 423-500 bp932-943.gen @ 589-671 bp 196-335 bp 501-632 bp 932-943.gen @ 759-899 bp12 bp Variably spliced 337-586 bp 633-883 bp 901-912.gen @ 787-1037 bp587-730 bp 884-1026 bp 910-915.gen @ 1134-1278 bp M146L mutation 731-795bp 1027-1092 bp 925-913.gen @ 413-578 bp H163R mutation 796-1017 bp1093-1314 bp 849-892.gen @ 336-558 bp A246E mutation 1018-1116 bp1315-1413 bp 951-952.gen @ 312-412 bp L286V mutation, variable spl1117-1204 bp 1414-1501 bp 983-1011.gen @ 61-149 bp 1205-1377 bp1502-1674 bp 874-984.gen @ 452-625 bp 1378-1497 bp 1674-1794 bp885-1012.gen @ 431-550 bp C410Y mutation 1493-2760 bp 1795-3060 bp930-919.gen @ −10 bp-cnd last AA, STOP, 3′UTR

TABLE 7 MUTATIONS AND POLYMORPHISMS IN THE ARMP GENE Nucleotide # Aminoacid # in ARMP.UPD in ARMP.PRO Comment A−>C₆₈₄ Met146Leu Pathogenic,Unique to AD affected. A−>G₇₃₆ His163Arg C−>A₉₈₅ Ala246Glu Pathogenic,Unique to AD affected. C−>T₁₀₂₇ Ala260Val Pathogenic, Unique to ADaffected. C−>T₁₁₀₂ Ala285Val Pathogenic, Unique to AD affected. C−>G₁₁₀₄Leu286Val Pathogenic, Unique to AD affected. C−>G₁₄₂₂ Leu392ValPathogenic, Unique to AD affected. G−>A₁₄₇₇ Cys410Tyr Pathogenic, Uniqueto AD affected. G−>T₈₆₃ Phe205Leu Polymorphism in normals C−>A₁₇₀₀non-coding 3′UTR polymorphism G−>A₂₆₀₁ non-coding 3′UTR polymorphismdelC₂₆₂₀ non-coding 3′UTR polymorphism

1. A method for detecting the presence of a mutant mammalian Presenilinprotein in a biological sample, the method comprising: (i) contactingthe biological sample with an isolated antibody selected from: (a) anisolated antibody that specifically binds to a mutant mammalianPresenilin protein, wherein the mutant mammalian Presenilin protein hasa mutation relative to a wild-type mammalian Presenilin, the mutationbeing selected from the group consisting of: M 146 L; H 163 R; A 246 E;L 286 V; and C 410 Y, and wherein the wild-type mammalian Presenilin hasthe amino acid sequence depicted in SEQ ID NO: 2 or 134; or (b) anisolated antibody raised against a mutant mammalian Presenilin protein,wherein the mutant mammalian Presenilin protein has a mutation relativeto a wild-type mammalian Presenilin, the mutation being selected fromthe group consisting of: M 146 L; H 163 R; A 246 E; L 286 V; and C 410Y, wherein the wild-type mammalian Presenilin has the amino acidsequence depicted in SEQ ID NO: 2 or 134, and wherein said isolatedantibody specifically binds to said mutant mammalian Presenilin protein;(ii) incubating the sample and the antibody under conditions sufficientto induce binding of the antibody to the mutant mammalian Presenilin inthe sample to form a complex; and (iii) detecting the complex.
 2. Amethod for detecting the presence of a mutant mammalian Presenilinprotein in a biological sample, the method comprising: (i) contactingthe biological sample with an isolated antibody selected from: (a) anisolated antibody that specifically binds to a mutant mammalianPresenilin protein, wherein the mutant mammalian Presenilin protein hasa mutation relative to a wild-type mammalian Presenilin, the mutationbeing selected from the group consisting of: A 260 V; A 285 V; and L 392V, and wherein the wild-type mammalian Presenilin has the amino acidsequence depicted in SEQ ID NO: 2 or 134; or (b) an isolated antibodyraised against a mutant mammalian Presenilin protein, wherein the mutantmammalian Presenilin protein has a mutation relative to a wild-typemammalian Presenilin, the mutation being selected from the groupconsisting of: A 260 V; A 285 V; and L 392 V, wherein the wild-typemammalian Presenilin has the amino acid sequence depicted in SEQ ID NO:2 or 134, and wherein said isolated antibody specifically binds to saidmutant mammalian Presenilin protein; (ii) incubating the sample and theantibody under conditions sufficient to induce binding of the antibodyto the mutant mammalian Presenilin in the sample to form a complex; and(iii) detecting the complex.
 3. The method according to claim 1 or 2,wherein said isolated antibody specifically binds to an epitopecomprising one of said mutations in said protein.
 4. The methodaccording to claim 1 or 2, wherein the antibody is a monoclonalantibody.
 5. The method according to claim 1 or 2, wherein the antibodyraised against the mutant mammalian Presenilin protein is a polyclonalantibody.
 6. The method according to claim 3, wherein the antibody is amonoclonal antibody.
 7. The method according to claim 3, wherein theantibody raised against the mutant mammalian Presenilin protein is apolyclonal antibody.
 8. A method for assessing the risk of Alzheimer'sDisease in a subject, comprising: (i) determining the level of a mutantmammalian Presenilin protein in a biological sample from a subject by:(a) contacting the biological sample with an isolated antibody selectedfrom: (1) an isolated antibody that specifically binds to the mutantmammalian Presenilin protein, wherein the mutant mammalian Presenilinprotein has a mutation relative to a wild-type mammalian Presenilin, themutation being selected from the group consisting of M 146 L; H 163 R; A246 E; L 286 V; and C 410 Y, and wherein the wild-type mammalianPresenilin has the amino acid sequence depicted in SEQ ID NO: 2 or 134;or (2) an isolated antibody raised against a mutant mammalian Presenilinprotein, wherein the mutant mammalian Presenilin protein has a mutationrelative to a wild-type mammalian Presenilin, the mutation beingselected from the group consisting of M 146 L; H 163 R; A 246 E; L 286V; and C 410 Y, wherein the wild-type mammalian Presenilin has the aminoacid sequence depicted in SEQ ID NO: 2 or 134, and wherein said isolatedantibody specifically binds to said mutant mammalian Presenilin protein;(b) incubating the sample and the antibody under conditions sufficientto induce binding of the antibody to the mutant mammalian Presenilin inthe sample to form a complex; (c) detecting the complex; and (ii)comparing the level of mutant mammalian Presenilin protein in thebiological sample from the subject to a normal level of mutant mammalianPresenilin protein, the normal level being determined from an average ofthe level of mutant mammalian Presenilin protein in a biological samplefrom a population consisting of subjects who do not show any symptoms ofAlzheimer's Disease, wherein a higher level of the protein in thebiological sample from the subject indicates a risk of Alzheimer'sDisease.
 9. A method for assessing the risk of Alzheimer's Disease in asubject, comprising: (i) determining the level of a mutant mammalianPresenilin protein in a biological sample from a subject by: (a)contacting the biological sample with an isolated antibody selectedfrom: (1) an isolated antibody that specifically binds to a mutantmammalian Presenilin protein, wherein the mutant mammalian Presenilinprotein has a mutation relative to a wild-type mammalian Presenilin, themutation being selected from the group consisting of: A 260 V; A 285 V;and L 392 V, and wherein the wild-type mammalian Presenilin has theamino acid sequence depicted in SEQ ID NO: 2 or 134; or (2) an isolatedantibody raised against a mutant mammalian Presenilin protein, whereinthe mutant mammalian Presenilin protein has a mutation relative to awild-type mammalian Presenilin, the mutation being selected from thegroup consisting of A 260 V; A 285 V; and L 392 V, wherein the wild-typemammalian Presenilin has the amino acid sequence depicted in SEQ ID NO:2 or 134, and wherein said isolated antibody specifically binds to saidmutant mammalian Presenilin protein; (b) incubating the sample and theantibody under conditions sufficient to induce binding of the antibodyto the mutant mammalian Presenilin in the sample to form a complex; (c)detecting the complex; and (ii) comparing the level of mutant mammalianPresenilin protein in the biological sample from the subject to a normallevel of mutant mammalian Presenilin protein, the normal level beingdetermined from an average of the level of mutant mammalian Presenilinprotein in a biological sample from a population consisting of subjectswho do not show any symptoms of Alzheimer's Disease, wherein a higherlevel of the protein in the biological sample from the subject indicatesa risk of Alzheimer's Disease.
 10. The method according to claim 8 or 9,wherein said isolated antibody specifically binds to an epitopecomprising one of said mutations in said protein.
 11. The methodaccording to claim 1 or 2, wherein the antibody is a monoclonalantibody.
 12. The method according to claim 1 or 2, wherein the antibodyraised against the mutant mammalian Presenilin protein is a polyclonalantibody.
 13. The method according to claim 10, wherein the antibody isa monoclonal antibody.
 14. The method according to claim 10, wherein theantibody raised against the mutant mammalian Presenilin protein is apolyclonal antibody.